National Medical Policy

National Medical Policy Subject: Policy Number: Cardiac Risk Assessment-Laboratory Tests NMP203 Effective Date*: February 2005 Updated: October 2015 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State s Medicaid manual(s), publication(s), citation(s), and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link X National Coverage Determination (NCD) Lipid Testing (190.23): http://www.cms.gov/medicare-coveragedatabase/search/advanced-search.aspx National Coverage Manual Citation X Local Coverage Determination (LCD)* B-Type Natriuretic Peptide (BNP) Testing: High Sensitivity C-Reactive Protein (hscrp) Testing: Non-Covered Services: http://www.cms.gov/medicare-coveragedatabase/search/advanced-search.aspx Article (Local)* X Other Palmetto GBA. MolDX. Corus CAD Test Coding and Billing Guidelines (M0009). http://www.palmettogba.com/palmetto/moldx.n sf/docscat/moldx%20website~moldx~browse %20By%20Topic~Covered%20Tests~8WXQ5R5 416?open&navmenu=%7C%7C Palmetto GBA. Jurisdiction 11 Part B. CMS National Laboratory Coverage Determinations: http://www.palmettogba.com/palmetto/provider s.nsf/docscat/providers~jurisdiction%2011%20 Part%20B~Browse%20by%20Topic~Lab?open& Cardiac Risk Assessment-Laboratory Tests Oct 15 1

Start=1&Count=50&Pg=1&navmenu=Browse% 5Eby%5ETopic%7C%7C Medicare Learning Matters Network. MLN Matters Number: MM5457. Related CR Release Date: January 26, 2007: http://www.cms.gov/outreach-and- Education/Medicare-Learning-Network- MLN/MLNMattersArticles/downloads/MM5457.pdf Medicare National Coverage Determinations (NCD) Coding Policy Manual and Change Report. January 2013: http://www.cms.gov/medicare/coverage/covera gegeninfo/downloads/manual201301.pdf None Medicare Learning Matters Network. MLN Matters Number: 4328. Medicare Learning Matters Network. October 26, 2012: http://www.cms.gov/outreach-and- Education/Medicare-Learning-Network- MLN/MLNMattersArticles/downloads/mm4328.pd f Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement (Please see Health Net National Medical Policies on Carotid Intima-Media Thickness, and Pharmocogenetics) Health Net, Inc. considers high-sensitivity C-reactive protein (hs-crp) testing for the assessment of coronary artery disease (CAD) risk medically necessary when all of the following are met: 1. The patient has undergone previous noninvasive tests for cardiac risk stratification (e.g., stress EKG, exercise thallium, stress echocardiography, SPECT) and been found to be at intermediate risk (not high risk*) as evidenced by meeting all of the following: Cardiac Risk Assessment-Laboratory Tests Oct 15 2

LDL cholesterol levels between 100 to 130 mg/dl; and Patient has 2 or more coronary heart disease (CHD) major risk factors, such as: Hypertension (BP 140 mmhg or higher, or on antihypertensive medication) Low HDL cholesterol (< 40 mg/dl) Diabetes Family history of premature CHD (CHD in male first degree relative < 55 years; CHD in female first degree relative < 65 years) Age (men age 45 years or older; women age 55 years or older) Current cigarette smoking Global risk assessment using Framingham point scoring** reveals a 10 to 20% risk of CHD per 10 years, i.e., a Framingham risk score of 0-9; and * Note: hs-crp testing is not recommended in high risk patients because an impact on their treatment outcomes has not been shown. **References: 1. Estimate of 10-Year Risk for Coronary Heart Disease Framingham Point Scores. Accessed at: http://www.nhlbi.nih.gov/guidelines/cholesterol/risktbl.htm 2. Framingham Risk Assessment Tool for Estimating 10-year Risk of Developing Hard CHD (Myocardial Infarction and Coronary Death). Accessed at: http://www.nhlbi.nih.gov/guidelines/cholesterol/risk_tbl.htm Health Net Inc. considers any of the following tests investigational, to assess cardiac risk because the medical literature is inconclusive regarding the utility of these tests for screening, diagnosis or management of CHD: High-sensitivity C-reactive protein (hs-crp) as a screening test for the general population or for monitoring response to therapy Complete profiles of cardiac risk (e.g., Cardiarisk, AAL Reference Laboratories offers an Atherosclerosis Activity Evaluation, Great Smokies Diagnostic Labs offers a Comprehensive Cardiovascular Risk Profile, Profilir technology) Apolipoprotein A-I (apo AI) Apolipoprotein B (apo B) Apolipoprotein E (apo E) Lipoprotein remnants: intermediate density lipoproteins (IDL) and small density lipoproteins (NMR LipoProfile). High density lipoprotein (HDL) subclasses (LpAI, LpAI/AII and/or HDL3 and HDL2) Low density lipoprotein (LDL) subclasses (small and large LDL particles) Lipoprotein(a) enzyme immunoassay Cardiac Risk Assessment-Laboratory Tests Oct 15 3

Angiotensin gene (AGT) (CardiaRisk) Fibrinogen Lipoprotein-associated phospholipase A2 (Lp-PLA2) (PLAC) Noninvasive measurements of arterial elasticity by means of blood pressure waveforms (e.g., HDI PulseWave, CVProfilor) Post-challenge insulin, high Iron levels Serum uric acid VAP (Vertical Auto Profile) Cholesterol Test Corus CAD gene expression testing Measurement of B-type natriuretic peptides (BNP) Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures will be replaced by ICD-10 code sets. Health Net National Medical Policies will now include the preliminary ICD-10 codes in preparation for this transition. Please note that these may not be the final versions of the codes and that will not be accepted for billing or payment purposes until the October 1, 2015 implementation date. ICD-9 Codes (not all inclusive list) 272.0 Pure hypercholesterolemia 272.1 Pure hyperglyceridemia 272.2 Mixed hyperlipidemia 272.3 Hyperchylomicronemia 272.4 Other and unspecified hyperlipidemia 411.1 Intermediate coronary syndrome ICD-10 Codes E78.0- E78.9 Disorders of lipoprotein metabolism and other lipidemias I20.0- I20.9 Angina pectoris I24.0- I24.9 Acute ischemic heart disease I25.1- I25.10 Chronic ischemic heart disease I25.110- I25.119 Atherosclerotic heart disease of native coronary artery with angina pectoris I25.810- I25.9 Other forms of chronic ischemic heart disease Cardiac Risk Assessment-Laboratory Tests Oct 15 4

CPT Codes 82172 Apolipoprotein, each 83695 Lipoprotein (a) 83698 Lipoprotein-associated phosolipase A2 (Lp-PLA) 83700 Lipoprotein, Blood; Electrophoretic Separation And Quantitation 83701 Lipoprotein, Blood; High Resolution Fractionation And Quantitation Of Lipoproteins Including Lipoprotein Subclasses When Performed (Eg, Electrophoresis, Ultracentrifugation) 83704 Lipoprotein, Blood; Quantitation Of Lipoprotein Particle Numbers And Lipoprotein Particle Subclasses (Eg, By Nuclear Magnetic Resonance Spectroscopy 83718 Lipoprotein, direct measurement; high density cholesterol (HDL cholesterol) 83721 LDL cholesterol 83880 Natriuretic peptide 84999 Unlisted chemistry procedure 86140 C-reactive protein 86141 C-reactive protein; high sensitivity (hscrp) 93799 Unlisted cardiovascular service or procedure 0311T Non-invasive calculation and analysis of central arterial pressure waveforms with interpretation and report HCPCS Codes N/A Scientific Rationale Update October 2015 The 2014 Update of the 2007 U.S. Preventive Services Task Force (USPSTF) recommendation on screening for carotid artery stenosis, has been completed. The USPSTF commissioned a systematic review to synthesize the evidence on the accuracy of screening tests, externally validated risk-stratification tools, the benefits of treatment of asymptomatic carotid artery stenosis with carotid endarterectomy (CEA) or carotid angioplasty and stenting (CAAS), the benefits from medications added to current standard medical therapy, and the harms of screening and treatment with CEA or CAAS. This recommendation applies to adults without a history of transient ischemic attack, stroke, or other neurologic signs or symptoms. The USPSTF recommends against screening for asymptomatic carotid artery stenosis in the general adult population. (D recommendation) The U.S. Preventive Services Task Force (USPSTF) makes recommendations about the effectiveness of specific preventive care services for patients without related signs or symptoms. It bases its recommendations on the evidence of both the benefits and harms of the service and an assessment of the balance. The USPSTF does not consider the costs of providing a service in this assessment. The USPSTF recognizes that clinical decisions involve more considerations than evidence alone. Clinicians should understand the evidence but individualize decision making to the specific patient or situation. Similarly, the USPSTF notes that policy and coverage decisions involve considerations in addition to the evidence of clinical benefits and harms. In the most recently published 2013 American College of Cardiology (ACC)/American Heart Association (AHA) Guideline on the Treatment of Blood Cholesterol to Reduce Cardiac Risk Assessment-Laboratory Tests Oct 15 5

Atherosclerotic Cardiovascular Risk in Adults, there is no recommendation of particle size in assessing risk or efficacy of treatments (Stone, 2014). Validated tools for linking levels of small dense LDL to clinical decision making, both in risk assessment and treatment response, are currently not available. Published data are inadequate to determine how such measurements should guide treatment decisions and whether these treatment decisions result in beneficial clinical outcomes. Other associated lipid parameters, such as triglycerides and HDL levels may be more useful than LDL size in assessing risk and treatment response. In the 2013 publication of the ACC/AHA Guideline on the Treatment of Blood Cholesterol to Reduce Atherosclerotic Cardiovascular Risk in Adults (Stone, 2013), only LDL-C, HDL-C and triglycerides are recommended as serum markers for assessing risk and managing disease. The guideline includes statements on treatments proven to reduce ASCVD events and do not include recommendations that include advanced lipoprotein testing. The guideline includes a critical question for future guidelines to determine "Whether on-treatment markers such as Apo B, Lp(a), or LDL particles are useful for guiding treatment decisions. Therefore, clinical application of these alternative serum markers for CVD risk remains undefined. In addition to the above guidelines, several meta-analyses, reviews and clinical studies have been published assessing the association of lipoprotein with CVD risk and events. Although consistently strong associations have been found between levels of lipoproteins and adverse cardiac health outcomes, evidence presented on the clinical utility of these measures continues to be inconsistent, conflicting and thus inconclusive. Clinical studies are ongoing to further assess and define the role and clinical utility of lipoprotein testing in CVD risk assessment, treatment and management. National Institute of Health Care Excellence (NICE, July 2014) has guidelines (CG181) on Lipid modification: cardiovascular risk assessment and the modification of blood lipids for the primary and secondary prevention of cardiovascular disease. However, these guidelines do not mention the laboratory testing for cardiac risks as noted in the policy statement. Studies Jonas et al. (2014) Approximately 10% of ischemic strokes are caused by carotid artery stenosis (CAS). Estimated prevalence of asymptomatic CAS is 1%. The purpose of this study was to evaluate evidence on screening and treating asymptomatic adults for CAS. The Study selection included good- or fair-quality trials of screening, carotid endarterectomy (CEA), or stenting compared with medical therapy or of intensification of medical therapy; systematic reviews; multi-institution studies reporting harms; and externally validated risk-stratification tools. No trials compared screening with no screening or stenting with medical therapy or assessed intensification of medical therapy, and no externally validated, reliable riskstratification tools were found. Given the specificity of ultrasonography (range, 88% to 94% for CAS 50% to 70%), its use in low-prevalence populations would yield many false-positive results. Absolute reduction of non-perioperative strokes was 5.5% (95% CI, 3.9% to 7.0%; 3 trials; 5223 participants) over approximately 5 years for CEA compared with medical therapy. The 30-day rates of stroke or death after CEA in trials and cohort studies were 2.4% (CI, 1.7% to 3.1%; 6 trials; 3435 participants) and 3.3% (CI, 2.7% to 3.9%; 7 studies; 17474 participants), respectively. Other harms of interventions included myocardial infarction, nerve injury, and hematoma. Trials may have overestimated benefits and used highly Cardiac Risk Assessment-Laboratory Tests Oct 15 6

selected surgeons. Medical therapy used in trials was outdated, and stroke rates have declined in recent decades. Harms may have been underreported. Current evidence does not establish incremental overall benefit of CEA, stenting, or intensification of medical therapy. Potential for overall benefit is limited by low prevalence and harms. Current evidence does not establish incremental overall benefit of CEA, stenting, or intensification of medical therapy. Potential for overall benefit is limited by low prevalence and harms. PRIMARY FUNDING SOURCE: Agency for Healthcare Research and Quality. Classification of risk of ischemic stroke is important for medical care and public health reasons. Whether addition of biomarkers adds to predictive power of the Framingham Stroke Risk or other traditional risk factors has not been studied in older women. Wasserheil-Smoller et al. (2014) completed a a case-control study of blood biomarkers, assayed in 972 ischemic stroke cases and 972 controls, nested in the Women's Health Initiative Observational Study of 93, 676 postmenopausal women followed for an average of eight-years. This study known as The Hormones and Biomarkers Predicting Stroke Study evaluated additive predictive value of two commercially available biomarkers: C-reactive protein and lipoprotein-associated phospholipase A2 to determine if they added to risk prediction by the Framingham Stroke Risk Score or by traditional risk factors, which included lipids and other variables not included in the Framingham Stroke Risk Score. As measures of additive predictive value, the C-statistic was used, a net reclassification improvement, category-less net reclassification improvement, and integrated discrimination improvement index. Addition of C-reactive protein to Framingham risk models or additional traditional risk factors overall modestly improved prediction of ischemic stroke and resulted in overall net reclassification improvement of 6 3%, (case net reclassification improvement=3 9%, control net reclassification improvement =2 4%). In particular, high-sensitivity C-reactive protein was useful in prediction of cardioembolic strokes (net reclassification improvement=12 0%; 95% confidence interval 4 3-19 6%) and in strokes occurring in less than three-years (net reclassification improvement=7 9%, 95% confidence interval 0 8-14 9%). Lipoprotein-associated phospholipase A2 was useful in risk prediction of large artery strokes (net reclassification improvement=19 8%, 95% confidence interval 7 4-32 1%) and in early strokes (net reclassification improvement=5 8%, 95% confidence interval 0 4-11 2%). C-reactive protein and lipoprotein-associated phospholipase A2 can improve prediction of certain subtypes of ischemic stroke in older women, over the Framingham stroke risk model and traditional risk factors, and may help to guide surveillance and treatment of women at risk. Additional peerreviewed studies are necessary. Kara et al. (2014) evaluated serum high-sensitivity C-reactive protein (hs-crp) and lipoprotein-related phospholipase A2 (Lp-PLA2) in patients who had acute ischemic stroke. In 200 patients who presented to an emergency service (i.e., acute ischemic stroke, 102 patients; control with no stroke, 98 patients), stroke patients were evaluated with the Canadian neurological scale and diffusion-weighted magnetic resonance imaging, and all patients were evaluated with the Glasgow coma scale and their serum hs-crp level and Lp-PLA2 activity were assessed. The volume of stroke lesions was calculated from magnetic resonance images. Patients who had stroke had higher mean serum hs-crp level (stroke, 7±6 mg/dl; control, mean ± standard deviation 1±1 mg/dl; P 0.001) and Lp-PLA2 activity (stroke, mean ± standard deviation 113±86 nmol/min/ml; control, mean ± standard deviation 103±50 Cardiac Risk Assessment-Laboratory Tests Oct 15 7

nmol/min/ml; P 0.001) than control patients who did not have stroke. The mean hs-crp level and Lp-PLA2 activity were higher in patients who had greater stroke severity (lower Canadian neurological scale score) and were higher in patients who had larger volume strokes. Higher hs-crp level and Lp-PLA2 activity are significantly associated with more severe neurologic impairment and larger infarct size in patients who have acute ischemic stroke. These biomarkers may be useful for rapid diagnosis and prediction of ischemic tissue volume in the early stage of ischemic stroke. These findings may be important for health care facilities that have limited access to emergency computed tomography scanning for the diagnosis of stroke. Lipoprotein-associated phospholipase A2 (LpPLA2) levels are associated with stroke, though whether this extends to all populations and stroke subtypes is unknown. Katan et al. (2014) Serum samples from stroke-free community participants in the Northern Manhattan Study were assayed for LpPLA2 mass and activity. Participants were followed annually for stroke. Cox-proportional-hazard models were fitted to estimate hazard-ratios and 95% confidence intervals (HR, 95% CI) for the association of LpPLA2 levels with ischemic stroke (IS), after adjusting for demographic and medical risk factors. Serum samples were available in 1946 participants, of whom 151 (7.8%) experienced a first IS during median follow-up 11 years. Mean age was 69 (SD 10), 35.6% were men, 20% non-hispanic Whites, 22% non-hispanic Blacks, and 55% Hispanics. LpPLA2 mass and activity levels were not associated with overall IS risk. LpPLA2 mass but not activity levels were associated with strokes due to large artery atherosclerosis (LAA; adjusted HR per SD 1.55, 95% CI 1.17-2.04). There was a dose-response relationship with LAA (compared to first quartile, 2nd quartile HR=1.43, 95% CI 0.23-8.64; 3rd quartile HR=4.47, 95% CI 0.93-21.54; 4th quartile HR=5.07, 95% CI 1.07-24.06). The associations between LpPLA2-mass and LAA-stroke risk differed by race-ethnicity (p=0.01); LpPLA2-mass was associated with increased risk of LAA among non-hispanic Whites (adjusted HR per SD 1.44, 95% CI 0.98-2.11), but not other race-ethnic groups. CONCLUSION: LpPLA2-mass levels were associated with risk of atherosclerotic stroke among non- Hispanic White participants, but not in other race-ethnic groups in the cohort. Further study is needed to confirm these race-ethnic differences and the reasons for them. Scientific Rationale Update October 2014 O'Donoghue et al. ( J Am Coll Cardiol. 2014) completed a study to assess the prognostic utility of lipoprotein(a) [Lp(a)] in individuals with coronary artery disease (CAD). Data regarding an association between Lp(a) and cardiovascular (CV) risk in secondary prevention populations are sparse. Plasma Lp(a) was measured in 6,708 subjects with CAD from 3 studies; data were then combined with 8 previously published studies for a total of 18,978 subjects. Across the 3 studies, increasing levels of Lp(a) were not associated with the risk of CV events when modeled as a continuous variable (odds ratio [OR]: 1.03 per log-transformed SD, 95% confidence interval [CI]: 0.96 to 1.11) or by quintile (Q5:Q1 OR: 1.05, 95% CI: 0.83 to 1.34). When data were combined with previously published studies of Lp(a) in secondary prevention, subjects with Lp(a) levels in the highest quantile were at increased risk of CV events (OR: 1.40, 95% CI: 1.15 to 1.71), but with significant between-study heterogeneity (p = 0.001). When stratified on the basis of low-density lipoprotein (LDL) cholesterol, the association between Lp(a) and CV events was significant in studies in which average LDL cholesterol was 130 mg/dl (OR: 1.46, 95% CI: 1.23 to 1.73, p < 0.001), whereas this relationship did not achieve statistical significance for studies with an average LDL cholesterol <130 mg/dl (OR: 1.20, 95% CI: 0.90 to Cardiac Risk Assessment-Laboratory Tests Oct 15 8

1.60, p = 0.21). Lp(a) is significantly associated with the risk of CV events in patients with established CAD; however, there exists marked heterogeneity across trials. In particular, the prognostic value of Lp(a) in patients with low cholesterol levels remains unclear. Patel et al. (2014) completed a study in which the purpose was to compare the association between variants at the chromosome 9p21 locus (Ch9p21) and risk of first versus subsequent coronary heart disease (CHD) events through systematic review and meta-analysis. Ch9p21 is a recognized risk factor for a first CHD event. However, its association with risk of subsequent events in patients with established CHD is less clear. The authors searched PubMed and EMBASE for prospective studies reporting association of Ch9p21 with incident CHD events and extracted information on cohort type (individuals without prior CHD or individuals with established CHD) and effect estimates for risk of events. The authors identified 31 cohorts reporting on 193,372 individuals. Among the 16 cohorts of individuals without prior CHD (n = 168,209), there were 15,664 first CHD events. Ch9p21 was associated with a pooled hazard ratio (HR) of a first event of 1.19 (95% confidence interval: 1.17 to 1.22) per risk allele. In individuals with established CHD (n = 25,163), there were 4,436 subsequent events providing >99% and 91% power to detect a per-allele HR of 1.19 or 1.10, respectively. The pooled HR for subsequent events was 1.01 (95% confidence interval: 0.97 to 1.06) per risk allele. There was strong evidence of heterogeneity between the effect estimates for first and subsequent events (p value for heterogeneity = 5.6 10(-11)). We found no evidence for biases to account for these findings. Ch9p21 shows differential association with risk of first versus subsequent CHD events. This has implications for genetic risk prediction in patients with established CHD and for mechanistic understanding of how Ch9p21 influences risk of CHD. Scientific Rationale Update October 2013 The determination of the underlying etiology of symptoms suggestive of obstructive coronary artery disease (CAD), 50% stenosis in a major coronary artery, is a common clinical challenge. Usual care in low to medium risk patients often involves a family history, risk factor assessment, and then stress testing with or without noninvasive imaging. If positive, this is often followed by invasive coronary angiography (ICA). Despite extensive adoption of this usual protocol, more than 60% of patients referred for angiography do not have obstructive CAD. In order to try to identify those symptomatic patients without obstructive CAD, who can avoid subsequent cardiac testing and look elsewhere for the cause of their symptoms, a whole blood gene expression score, or Corus CAD test is being proposed. A Policy Statement from the American Heart Association (2012) discussed the role of genetics and cardiovascular disease treatment and diagnosis but did not address gene expression as is measured in the Corus CAD test. McPherson et al. (2013) completed the Clinical Trial on The IMPACT-Cardiology (i.e., Investigation of a Molecular Personalized Coronary Gene Expression Test on Cardiology Practice Pattern, which was done to assess the impact of Corus CAD use on clinical decision-making during the assessment of stable chest pain patients in the cardiology setting. The Clinical Trials.gov identifier is ClinicalTrials.gov Identifier: NCT01251302 and it was last updated in February 2013. The study included a prospective cohort of 83 patients eligible for analysis. These patients were referred to six cardiologists for evaluation of suspected CAD and were matched by clinical factors to patients in a historical cohort. The study protocol was designed to Cardiac Risk Assessment-Laboratory Tests Oct 15 9

evaluate and compare the cardiologists diagnostic strategies before and after receiving the Corus CAD results for their patients. The pre-corus CAD assessment of patients CAD probability noted their preliminary management decision. This pre- Corus CAD assessment was compared to physicians assessment of CAD probability after The IMPACT-Cardiology protocol and this was designed to evaluate and compare the cardiologists diagnostic strategies before and after receiving the Corus CAD results for their patients. In this study following communication of Corus CAD results, a change in diagnostic testing (e.g. myocardial perfusion imaging, CTA and cardiac catheterization) was noted in 48 patients [58%, 95% CI (46%, 69%)]. More patients had a decreased versus increased level of testing (n=32 (39%) vs n=16 (19%), p=0.03). In particular, 91% (29 of 32) of patients with decreased testing had low Corus CAD ( 15), while 100% (16 of 16) of patients with increased testing had elevated Corus CAD (p<0.001). The most common change was among patients considered for referral to non-invasive imaging or invasive angiography prior to the Corus CAD test who were then referred to either no intervention or medical management after receiving a low Corus CAD score. In addition, none of the patients with low scores ( 15) saw an increase in testing. The IMPACT-CARD trial demonstrated that among patients with a low Corus CAD score, the management decisions led to a decrease in non-invasive cardiac imaging and invasive angiography. The study was sponsored by CardioDx. This study is limited by comparison with historical controls, which were not well-matched to the study population. In addition, the impact of management changes in this study is uncertain. There is no information provided on whether the management changes led to beneficial effects on outcome, and it is not possible to estimate the likelihood of benefit from the information given in this study. Therefore, it is not possible to conclude that the GES score leads to changes in management that improve outcomes. The Clinical Trial Personalized Risk Evaluation and Diagnosis In the Coronary Tree (PREDICT), with the ClinicalTrials.gov identifier NCT00500617 was terminated in 2012. The Clinical Trial on Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS) has been completed, with the CardioDxClinicalTrials.gov Identifier of NCT01117506. The sponsor of this study is CardioDx. The COMPASS study evaluated test performance in symptomatic patients (N=431) referred for myocardial perfusion imaging (MPI), a procedure used prior to angiography. The gold standard is a combination of either invasive angiography or CT-angiography, both determined in core laboratories, so that all patients independent of their MPI results, had gold standard data on their coronary anatomy. Obstructive CAD prevalence was only 15%. Positive MPI scans were seen in 11% of patients The primary endpoint of the area under the receiver-operating characteristics curve (AUC for ROC) analysis for discriminating patients with and without obstructive CAD (50% stenosis by quantitative angiography or core-lab CT-angiography) yielded AUCs of 0.79, (p<0.001). In the COMPASS study, the overall accuracy of the GES score in predicting cardiac events was superior to MPI in patients who were referred for MPI testing. However, in this study, the reported sensitivity of MPI was considerably lower than generally reported in the literature. Also, it is unclear from the COMPASS study whether patients with a positive MPI could safely forego further testing based on a low GES score. There is another Clinical Trial on A Registry to Evaluate Patterns of Care Associated with the Use of Corus CAD in Real World Clinical Care Settings (PRESET) with the Cardiac Risk Assessment-Laboratory Tests Oct 15 10

ClinicalTrials.gov Identifier of NCT01677156. This was last updated on August 29 2012, and is currently recruiting participants. Though the diagnostic properties of Corus CAD have been evaluated in previous observational studies, there are limited data regarding how primary care clinicians are using the results of the test in the care and management of patients with angina. This registry is designed to examine the relationship between the Corus CAD score and patterns of care regarding diagnostic testing in real world clinical care settings. There is no estimated date of completion at this time. There is another Clinical Trial Primary Care Providers Use of a Gene Expression Test in Coronary Artery Disease Diagnosis (IMPACT-PCP) which is ongoing but not recruiting participants. The ClinicalTrials.gov Identifier is NCT01594411, and was last updated in February 2013. This is a prospective, multi-center study examining the clinical impact of the Corus CAD assay in approximately 250 evaluable subjects with no history of obstructive coronary artery disease who now present with chest pain or anginal-equivalent symptoms to a primary care physician (PCP) for evaluation. The primary completion date is listed as February 2013, but this study is not completed at this time. There is another Clinical Trial Investigation of a Novel Gene Expression Test for the Diagnosis of Obstructive Coronary Artery Disease on Physician's Practice Pattern which is currently recruiting participants. The ClinicalTrials.gov Identifier is NCT01557855 and it was last verified in February 2013. The objective of this study is to collect data on the commercial use of CORUS (validated quantitative in vitro diagnostic test) Coronary Artery Disease (CAD) blood test to evaluate the clinical referral patterns of Primary Care Physicians after receipt of their patients' CORUS Score, and to better understand patient management patterns for clinicians ordering the test. The estimated primary completion date is March 2013. Although the results of the evaluation of the Corus CAD test are promising, its results should be interpreted carefully as patients with diabetes mellitus and chronic inflammatory or autoimmune disorders were excluded from test development and validation. Furthermore, this test was derived and tested in predominantly Caucasian patient populations. Given the known variations in the prevalence of CAD in different ethnic/racial backgrounds, results of this test in non-caucasian populations should be interpreted with caution. In summary, the data regarding Corus CAD are limited in reaching conclusions about its performance and value in patients being evaluated for obstructive CAD. While the available evidence suggests the test has a moderately high negative predictive value, additional evidence supporting the analytical validity, clinical validity, and clinical utility is necessary. Two of the Clinical Trials noted within this Scientific Rationale (i.e., IMPACT and COMPASS) were just completed this year (2013). Additional research is necessary to determine if a low CAD score (i.e., GES Score) and changes in the individual s care management leads to better long-term outcomes for the individual. It was also unclear from the COMPASS study if a positive myocardial perfusion imaging (MPI) could safely forego further testing based on a low GES score. Scientific Rationale Update June 2013 The ACCF/AHA Guideline for Assessment of Cardiovascular Risk in Asymptomatic Adults, has not been updated since 2010. Cardiac Risk Assessment-Laboratory Tests Oct 15 11

The American Heart Association/American Stroke Association Council recommendations were last updated in 2011. The guideline states that measurement of inflammatory markers such as Lp-PLA2 in patients without cardiovascular disease may be considered to identify patients who may be at increased risk of stroke, although their effectiveness (i.e. usefulness in routine clinical practice) is not well established. Additional well-designed clinical trials are necessary to establish the clinical utility of Lp-PLA2 for cardiovascular risk assessment and to determine the role of Lp-PLA2 as a potential adjunct to traditional risk assessment in the overall management of stroke in adults. The European Guidelines on Cardiovascular Disease Prevention in Clinical Practice were updated in 2012. Fibrinogen may be measured as part of refined risk assessment in patients with an unusual or moderate CVD risk profile. Recommendation was IIb B Weak (Fibrinogen should not be measured in asymptomatic low-risk individuals and high-risk patients to assess 10-year risk of CVD. (European 2012). While advanced lipoprotein analysis detects elevated or reduced levels of lipoproteins, there is lack of agreement on how this information would be used in clinical decision-making. In addition, while the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III identifies lipoprotein (a) and apolipoprotein analysis as emerging technologies, it does not recommend their routine use in identifying persons at risk for cardiovascular disease or ischemic stroke. Some experts continue to argue that apo B is superior to LDL cholesterol, and that the apo B/apo A-I ratio is superior to the LDL/HDL ratio, as predictors of cardiovascular risk, and that these apolipoprotein measures should supplement or replace traditional lipid measures. A publication from the American Diabetes Association and the American College of Cardiology Foundation (2009) included specific recommendations for incorporating apo B testing into clinical care for highrisk patients. This expert panel stated, ApoB and LDL particle number also appear to be more discriminating measures of the adequacy of LDL lowering therapy than are LDL cholesterol or non-hdl cholesterol. Therefore, they recommend that for patients with metabolic syndrome who are being treated with statins, both LDL cholesterol and apo B should be used as treatment targets, with an apo B target of less than 90mg/dl. Treatment should be intensified for patients with apob above this level even if target LDL has been achieved. However, the evidence suggests that any incremental improvement in predictive ability over traditional measures is likely to be small and of uncertain clinical significance. In addition, none of the major lipid treatment guidelines, such as The Adult Treatment Panel III (ATP III) of the National Cholesterol Education Program (NCEP), have yet to formally incorporate the measurement of apolipoproteins into their recommendations. This creates difficulties in interpreting and applying the results of apo B and/or apo B/apo A-I measurements to routine clinical care. Therefore, it does not appear likely that apolipoprotein measures will replace traditional lipid measurements for cardiovascular risk prediction in routine clinical care, at this time. A large body of research has focused on the correlation between lipid levels and the underlying apolipoprotein E (apo E) genotype. Other studies have focused on the relationship between genotype and clinical disease. Clinical evidence suggests that apo E is not clinically useful in providing additional information on risk for CAD when compared with other established and emerging risk factors. None of the studies reviewed provide adequate data to suggest that apo E genotype improves outcomes when used in clinical care. Cardiac Risk Assessment-Laboratory Tests Oct 15 12

Retrospective cross-sectional studies have suggested that the protective effect of HDL was associated primarily with the HDL-2 subclass. However, these studies could not determine whether decreased HDL-2 preceded the development of cardiovascular disease or was its result. A number of large, prospective studies designed to answer this question have reported mixed results. A nonprofit independent licensee of the BlueCross BlueShield Association, notes that subclassification information on risk assessment for CAD, has not been reported consistently in all studies. HDL subclassification has not been incorporate into quantitative risk assessment models or treatment guidelines, such as the Adult Treatment Panel (ATP III) that can be used in clinical practice. Intermediate density lipoprotein (remnant-like particles). An immunoseparation assay has received approval from the U.S. Food and Drug Administration (FDA) for the direct measurement of intermediate density lipoprotein. While measurement of intermediate-density lipoproteins (IDLs) has emerged as an important research tool in evaluating cardiac risk factors and understanding how different components of plasma triglycerides contribute to cardiac risk, it is unclear how the management of IDLs can be used in the management of the patient. The majority of publications focus on the pathophysiology and basic science aspects of IDL, with a smaller number of research studies reporting data with potential clinical relevance. There are no prospective, large-scale cohort studies that evaluated IDLs as a predictor of cardiovascular risk, nor are there any large diagnostic studies that evaluated the utility of IDLs in diagnosing type III hyperlipidemia. None of the available studies provide guidance on the clinical use of IDL measurements, nor does this evidence suggest that health outcomes are improved as a result of measuring IDL level. Low density (small) lipoprotein particle size. Small LDL size is one component of an atherogenic lipid profile that also includes increased triglycerides, increased apolipoprotein B, and decreased HDL. Some studies have reported that LDL size is an independent risk factor for CAD, and others have reported that a shift in LDL size may be useful marker of treatment response. A relatively small number of published articles contain clinically relevant evidence on the utility of measuring the concentration of small, dense LDL (or LDL particle size). The available publications primarily focus either on the use of these measures as a predictor of cardiovascular risk, or the effect of pharmacologic treatment on small, dense LDL. Studies predicting cardiac risk were cross-sectional studies that evaluated the association of small, dense LDL with a variety of cardiovascular outcomes. There are a paucity of large, prospective cohort studies that evaluate the predictive ability of small, dense LDL for future cardiovascular events. The studies of treatment effect examined the impact of diet and/or pharmacologic agents on small, dense LDL and other LDL subclasses. These studies generally confirmed that small, dense LDL is impacted preferentially by fibrate treatment, and possibly also by statin therapy. However, the studies do not demonstrate that preferentially targeting small, dense LDL leads to improved outcomes, as compared to using the standard LDL targets that are widespread in clinical care. These newly published studies do not provide evidence that measurement of small, dense LDL leads to improved clinical outcomes. Tools for linking concentration of LDL particles to clinical decision making, both in risk assessment and treatment response, are currently not available. Scientific Rationale Update September 2012 The Palmetta GBA site notes: "Palmetto GBA has completed the Corus CAD Gene Expression technical assessment and determined that the test meets criteria for analytical and clinical validity, and clinical utility as a reasonable and necessary Cardiac Risk Assessment-Laboratory Tests Oct 15 13

Medicare benefit. Effective January 1, 2012, Palmetto GBA will reimburse services for Corus CAD. To report a Corus CAD service, submit the following claim information: CPT code 84999 - Unlisted chemistry procedure". CardioDX has a press release dated August 8, 2012, which notes the following: "CardioDx, Inc., announced Palmetto GBA, a national contractor that administers Medicare benefits, has established coverage for the company s Corus CAD gene expression test for the evaluation of patients presenting with typical and atypical symptoms suggestive of coronary artery disease. Corus CAD is promoted to assess whether or not a stable non-diabetic patient s symptoms are due to obstructive coronary artery disease (CAD), enabling many patients to avoid unnecessary invasive testing and exposure to imaging-related radiation risks or imaging agent intolerance. The test has been validated in patient cohorts, including two prospective, multicenter U.S. trials, PREDICT and COMPASS. Per CardioDX, a retrospective, multicenter chart review study and the prospective IMPACT trial at Vanderbilt University demonstrated that Corus CAD use yielded significant and clinically relevant changes in patient management decisions in both primary care and cardiology settings. Corus CAD is the first gender-specific test for obstructive CAD accounting for critical biological differences between men and women. Corus CAD is not intended for use in patients who are diabetic, have been diagnosed with prior myocardial infarction (MI) or have had a previous revascularization procedure, or are currently taking steroids, immunosuppressive agents or chemotherapeutic agents. The Corus CAD test measures the RNA levels of 23 genes. Because peripheral blood cell RNA levels are altered when obstructive coronary artery disease is present, the Corus CAD score aids clinicians in assessing whether an individual patient s symptoms may be due to obstructive coronary artery disease. Effective 6/22/12 Revisions in Medicare Coverage: " Coverage for all Medicare members when one Medicare A/B Administrative Contractor (MAC) processes all of the claims for a particular Medicare covered item or service for all Medicare beneficiaries around the country. This generally occurs when there is only one supplier of a particular item, medical device or diagnostic test (e.g., certain pathology and lab tests furnished by independent laboratories). In this situation, Medicare Advantage (MA) plans must follow the coverage requirements OR LCD of the MAC that enrolled the supplier and processes all of the Medicare claims for that item, test or service". (Effective 6-22-12, Medicare CMS Manual, Transmittal 107, Chapter 90.2, Available at: http://www.cms.gov/regulations-and- Guidance/Guidance/Transmittals/Downloads/R107MCM.pdf Elashoff et al. (2011) Alterations in gene expression in peripheral blood cells have been shown to be sensitive to the presence and extent of CAD. A non-invasive blood test that could reliably assess obstructive CAD likelihood would have diagnostic utility. Microarray analysis of RNA samples from a 195 patient Duke CATHGEN registry case: control cohort yielded 2,438 genes with significant CAD association (p < 0.05), and identified the clinical/demographic factors with the largest effects on gene expression as age, sex, and diabetic status. RT-PCR analysis of 88 CAD classifier genes confirmed that diabetic status was the largest clinical factor affecting CAD associated gene expression changes. A second microarray cohort analysis limited to non-diabetics from the multi-center PREDICT study (198 patients; 99 case: control pairs matched for age and sex) evaluated gene expression, clinical, and cell population predictors of CAD and yielded 5,935 CAD genes (p < 0.05) with an intersection of 655 genes with the CATHGEN results. Biological pathway (gene ontology and literature) and statistical analyses (hierarchical clustering and logistic Cardiac Risk Assessment-Laboratory Tests Oct 15 14

regression) were used in combination to select 113 genes for RT-PCR analysis including CAD classifiers, cell-type specific markers, and normalization genes.rt-pcr analysis of these 113 genes in a PREDICT cohort of 640 non-diabetic subject samples was used for algorithm development. Gene expression correlations identified clusters of CAD classifier genes which were reduced to meta-genes using LASSO. The final classifier for assessment of obstructive CAD was derived by Ridge Regression and contained sex-specific age functions and 6 meta-gene terms, comprising 23 genes. This algorithm showed a cross-validated estimated AUC = 0.77 (95% CI 0.73-0.81) in ROC analysis. The authors have developed a whole blood classifier based on gene expression, age and sex for the assessment of obstructive CAD in non-diabetic patients from a combination of microarray and RT-PCR data derived from studies of patients clinically indicated for invasive angiography. Clinical Trial Registration Information: PREDICT, Personalized Risk Evaluation and Diagnosis in the Coronary Tree, NCT00500617. However, the Clinical Trial noted in the paragraph above, with the ID number of NCT00500617, was terminated and last updated on June 4, 2012. The purpose of this prospective, multi-center, observational study, was to develop and validate a diagnostic blood assay for atherosclerotic CAD. The Assay will use quantitative realtime PCR (RT-PCR) to quantify the expression of multiple genes from circulating peripheral blood cells to assess the presence of clinically significant CAD in a patient. The study was divided into four sequential segments with unique subjects and goals: gene discovery (segment 1), Assay development (segment 2), Assay validation (segment 3), and additional Assay testing (segment 4). The primary analysis will be performed during the third segment of the study using a subset of the enrolled subjects ("primary subjects"). Primary subjects will be defined by additional eligibility criteria beyond the general eligibility criteria required for enrollment in the overall study. The additional, post-enrollment eligibility criteria will be based on clinical and demographic factors that are found during the course of the study to affect the expression of genes used in the Assay. Such factors will be identified during gene discovery and algorithm development. The post-enrollment eligibility criteria will be defined prior to the beginning of the Assay validation segment of the study. In addition, three substudies are planned and will enroll up to 1500 subjects. The first substudy will include subjects undergoing cardiac CT angiography (CTA) and aims to determine how gene expression correlates with total coronary atheroma burden, as measured by CTA. The second substudy examines sample handling and shipping conditions and does not affect the treatment of the subjects. The third substudy will focus on additional algorithm development and validation in a non-diabetic female patient population. However, since this trial was terminated, it does not seem that any of the substudies or post studies would be done. There is another Clinical Trial on 'Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS)', also noted above, which is ongoing, but not recruiting participants. This was last updated on March 16, 2012 with the ClinicalTrials.gov Identifier of NCT01117506. The purpose of this trial was to validate the use of Corus CAD blood assay in subjects who are referred for the work-up of CAD. The study will evaluate the clinical utility of a gene expression test (Corus CAD) in subjects referred for myocardial perfusion imaging (MPI) work-up for suspected obstructive atherosclerotic coronary artery disease (CAD). The Corus CAD is a gene expression test that quantify the expression of multiple genes from circulating peripheral blood cells to detect the presence of clinically significant obstructive CAD in patients with chest pain. Cardiac Risk Assessment-Laboratory Tests Oct 15 15

A Clinical Trial on 'Investigation of a Molecular Personalized Coronary Gene Expression Test on Cardiology Practice Pattern (IMPACT-CARD), is also ongoing, but not recruiting participants. This was last updated on March 16, 2012 with the ClinicalTrials.gov Identifier of NCT01251302. The purpose of this study was to investigate whether the use of Corus CAD blood assay changes the diagnostic testing pattern in patients referred to a cardiologist for the evaluation of chest pain or anginal equivalent symptoms. Lansky et al. (American Heart Journal 2012) summarizes the PREDICT trial as noted above. It also notes that this test has not yet been studied to predict the risk of future cardiovascular events. In addition, beyond only a subset of the cohort having MPIs, there is a significant referral bias inherent in the analysis because patients with negative MPI were probably less likely to be referred to diagnostic catheterization, therefore impacting the reported negative predictive value of MPI. Irrespective of these limitations, the comparative validation study applies equally to both MPI and GES, and the positive predictive value should not be greatly impacted by the referral bias because patients with positive MPIs are likely to be referred to diagnostic catheterization given current practice patterns. It also notes that the use of a bloodbased GES may be particularly helpful in the assessment of obstructive CAD in nondiabetic patients and, in particular, women for whom the use of symptoms and functional testing has proven unreliable. However, further studies are needed to validate whether this test or other methods that use individualized genomic data will help promote more efficient and appropriate use of coronary angiography in women. (7/13/2002) Palmetto GBA has determined APOLIPOPROTEIN (Apo) E genotype testing, developed to assess the risk of cardiovascular disease, has insufficient evidence to support reasonable and necessary criteria for Medicare reimbursement. Therefore, Palmetto GBA will deny ApoE test services. Medicare does not cover High-sensitivity C-reactive protein (hscrp) testing as a screening test for the general population or for monitoring response to therapy. The clinical value for these uses has not been established. Homocysteine testing, the measurement of 12-hour fasting levels of homocysteine in plasma, can be used as part of an overall assessment of patient risk for cardiovascular disease (CVD). It has been suggested that homocysteine testing can provide information regarding CVD risk in addition to that provided by more common lipoprotein tests, such as total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. Homocysteine testing may also aid in treatment decisions, based on the assumption that there is a health benefit associated with lowering elevated homocysteine levels. Although there is evidence from epidemiologic studies for an association between high plasma homocysteine levels and increased cardiovascular disease (CVD) risk, not all prospective studies have supported such a relationship. In addition, there is no consensus on target levels or safe levels of total homocysteine, and there is a paucity of evidence in relation to women and racial minorities. Moreover, it remains to be proven whether reduction of plasma homocysteine levels has a positive effect on the risk or progression of CVD. Therefore, at the present time, there is insufficient evidence to support the use of homocysteine testing for screening, diagnosis, or management of CVD in healthy, atrisk, or diagnosed patient populations. The aim of the 2012 guidelines from the Fifth Joint Task Force (JTF) of the European Societies on Cardiovascular Disease Prevention in Clinical Practice is to give an Cardiac Risk Assessment-Laboratory Tests Oct 15 16

update of the present knowledge in preventive cardiology. The following has been noted: Because apob (the main apoprotein of atherogenic lipoproteins) levels have so frequently been measured in outcome studies in parallel with LDL cholesterol, apob can be substituted for LDL cholesterol, but it does not add further to the risk assessment. There is still not sufficient scientific evidence for any high density lipoprotein (HDL) cholesterol value to be considered as a goal of therapy, although HDL cholesterol,1.0 mmol/l (40 mg/dl) in men and,1.2 mmol/l (45 mg/dl) in women may be regarded as a marker of increased risk. Lipoprotein(a) is a low-density lipoprotein (LDL) to which is attached an additional protein called apolipoprotein(a). High concentrations of Lp(a) are associated with increased risk of CHD and ischaemic stroke, although there is no randomized intervention showing that reducing Lp(a) decreases CVD risk. There is no justification for screening the general population for Lp(a) at present, and no evidence that any value should be considered as a target. Apolipoprotein A1 (apoa1) is the major apoprotein of HDL. It is beyond doubt that the apob:apoa1 ratio is one of the strongest risk markers. However, it is still not established whether this variable should be used as a treatment goal. As the measurement of apolipoproteins is not available to all physicians in Europe, is more costly than currently used lipid variables, and does not add more information, its use is not as yet generally recommended. Fibrinogen should not be measured in asymptomatic low-risk individuals and high-risk patients to assess 10-year risk of CVD. High-sensitivity CRP has shown consistency across large prospective studies as a risk factor integrating multiple metabolic and low-grade inflammatory factors underlying the development of unstable atherosclerotic plaques, with a magnitude of effect matching that of classical major risk factors. This marker was used in individuals showing a moderate level of risk from clinical assessment of major CVD risk factors. However, several weak points exist when including this biomarker for highrisk assessment: 1. Multiplicity of confounders: dependence on other classical major risk factors. 2. Lack of precision: narrow diagnostic window for hscrp level and risk of CVD. 3. Lack of specificity: similar level of risk for other noncardiovascular causes of morbidity and mortality (e.g. other low-grade inflammatory diseases). 4. Lack of dose effect or causality relationship between changes in hscrp level and risk of CVD. 5. Lack of specific therapeutic strategies or agents targeting circulating CRP and showing reduction in CVD incidence. Statements numbered 1-5 above are also noted for fibrinogen. Homocysteine has shown precision as an independent risk factor for CVD. The magnitude of effect on risk is modest, and consistency is often lacking, mainly due to nutritional, metabolic (e.g. renal disease), and lifestyle confounders. In addition, intervention studies using B vitamins to Cardiac Risk Assessment-Laboratory Tests Oct 15 17

reduce plasma homocysteine have proven inefficient in reducing risk of CVD. LpPLA2 has recently emerged as a marker with high consistency and precision as an independent risk factor for plaque rupture and atherothrombotic events. The magnitude of effect on risk remains modest at the level of the general population; study limitations or bias are present. Studies Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a recently identified and potentially useful plasma biomarker for cardiovascular and atherosclerotic diseases. However, the correlation between the Lp-PLA2 activity and carotid atherosclerosis remains poorly investigated in patients with metabolic syndrome (MetS). Gong et al. completed a study aimed to evaluate the potential role of Lp-PLA2 as a comprehensive marker of metabolic syndrome in individuals with and without carotid atherosclerosis. The authors documented 118 consecutive patients with MetS and 70 age and sex matched healthy subjects served as controls. The patients were further divided into two groups: 39 with carotid plaques and 79 without carotid plaques to elucidate the influence of Lp-PLA2 on carotid atherosclerosis. The plasma Lp-PLA2 activity was measured by using ELISA method and carotid intimal-media thickness (IMT) was performed by ultrasound in all participants. Lp-PLA2 activity was significantly increased in MetS subgroups when compared with controls, and was higher in patients with carotid plaques than those without plaques (P < 0.05). significant difference was found in Lp-PLA2 that was obtained between patients with three and four disorders of metabolic syndrome (P < 0.01). Age (β = 0.183, P = 0.029), LDL-cholesterol (β = 0.401, P = 0.000) and waist-hip ratio (β = 0.410, P = 0.000) emerged as significant and independent determinants of Lp-PLA2 activity. Multiple stepwise regression analysis revealed that LDL-cholesterol (β = 0.309, P = 0.000), systolic blood pressure (β = 0.322, P = 0.002) and age (β = 0.235, P = 0.007) significantly correlated with max IMT, and Lp-PLA2 was not an independent predictor for carotid IMT. Lp-PLA2 may be a modulating factor for carotid IMT via age and LDL-cholesterol, but not an independent predictor in the pathophysiological process of carotid atherosclerosis in patients with MetS. Additional peer reviewed studies with long-term outcomes are necessary. Constantinides et al. (2011) completed a recent meta-analysis which showed that both plasma lipoprotein-associated phospholipase A(2) (Lp-PLA(2) ) mass and activity may independently predict cardiovascular events. Lp-PLA(2) activity but not mass was found to be a determinant of cardiovascular outcome in type 2 diabetes mellitus. We questioned whether relationships of carotid intima media thickness (IMT), a measure of subclinical atherosclerosis, with Lp-PLA(2) mass differ between diabetic and nondiabetic subjects. Relationships of IMT with plasma Lp-PLA(2) mass (turbidimetric immunoassay) were compared in 74 patients with type 2 diabetes and in 64 nondiabetic subjects. IMT was increased (P=0 016), but plasma Lp-PLA(2) mass was decreased in patients with diabetes compared to nondiabetic subjects (277±66 vs. 327±62ΜgL(-1), P<0 001). In nondiabetic subjects, IMT was correlated positively with Lp-PLA(2) (r=0 325, P<0 009); multiple linear regression analysis confirmed an independent association of IMT with Lp-PLA(2) (ß=0 192, P=0 048). In contrast, IMT was unrelated to Lp-PLA(2) in patients with diabetes (r=0 021, P=0 86), and the relationship of IMT with Lp-PLA(2) was different in diabetic and control subjects (P<0 001). The relationship of Lp-PLA(2) with the total cholesterol/high-density lipoprotein (HDL) cholesterol ratio also differed between diabetic and nondiabetic subjects (P<0 001). Plasma Lp-PLA(2) may relate to early Cardiac Risk Assessment-Laboratory Tests Oct 15 18

stages of atherosclerosis development. In diabetes mellitus, in contrast, the association of IMT with plasma Lp-PLA(2) mass is abolished, which could be partly attributed to redistribution of Lp-PLA(2) mass from apolipoprotein B-containing lipoproteins towards HDL. These findings raise questions about the usefulness of plasma Lp-PLA(2) mass measurement as a marker of subclinical atherosclerosis in type 2 diabetes mellitus. Additional peer-reviewed studies are necessary The American College of Cardiology (ACC), and the American Heart Association (AHA) Task Force recommendations on assessing cardiovascular risk in asymptomatic adults by (Greenland et al., 2010), have not been updated since this time. The American Heart Association, the American College of Cardiology and the National Cholesterol Education Program Adult Treatment Panel guidelines (ATP III) have not issued formal recommendations for many of these laboratory tests. Further peer-reviewed evidence based literature is needed to establish the assessment of risk factors in determining coronary artery disease. Scientific Rationale Update July 2012 Serum CRP Screening In a review of over 15,000 individuals from the third National Health and Nutrition Examination Survey (NHANES) in the United States, high serum CRP (>3 mg/l) was rare in the absence of any borderline or abnormal coronary risk factor (4.4 percent in men and 10.3 percent in women). The likelihood of a high serum CRP was largely attributable to the presence of other risk factors (78 percent in men and 67 percent in women). It was concluded that serum CRP may have limited clinical utility as a screening tool beyond other known cardiovascular risk factors. Similar findings were noted in the ARIC study, which assessed the association of 19 novel risk markers, including serum CRP, with incident CHD in nearly 16,000 adults followed for up to 15 years. The CRP level did not add significantly to the basic riskfactor model (age, sex, total and high-density lipoprotein cholesterol levels, systolic blood pressure, antihypertensive medication use, smoking status, and diabetes) as assessed by the change in area under receiver operating characteristic curves. In total, these studies suggest that, while serum CRP does appear to act as an independent predictor of cardiovascular disease, the predictive value added to that determined by screening for other coronary risk factors in the general population is small. Instead, serum CRP appears to add the greatest predictive value in a subset of patients with intermediate CHD risk as determined by other measures such as the Framingham Risk Score. Screening for Coronary Heart Disease Although screening appears to identify patients at increased risk, there is a paucity of evidence that such screening actually improves outcomes. Some data suggest that medical therapy or revascularization in selected patients with silent ischemia may improve outcomes. MRFIT and the Atenolol in Silent Ischemia Study demonstrated benefit from risk factor reduction and atenolol, respectively. A benefit from revascularization was suggested by results from the NIH-sponsored Asymptomatic Cardiac Ischemia Pilot (ACIP) trial, which randomly assigned 558 patients to one of three treatment strategies: angina-guided medical therapy; ischemia-guided medical therapy; or revascularization with coronary artery bypass graft (CABG) or percutaneous coronary intervention (PCI). At two years, total mortality was significantly lower with revascularization compared to ischemia-guided Cardiac Risk Assessment-Laboratory Tests Oct 15 19

or angina-guided medical therapy (1.1 versus 4.4 and 6.6 percent). The composite end point of death, MI, or recurrent cardiac hospitalization was also reduced. The only appropriate indication for revascularization in asymptomatic patients is to improve prognosis since revascularization cannot improve symptoms. The choice of revascularization and of the procedure is dependent upon coronary anatomy, left ventricular function, and the presence or absence of diabetes. Apoproteins (Apolipoproteins) Lipids, such as cholesterol and triglycerides, are insoluble in plasma and circulating lipid is carried in lipoproteins that transport the lipid to various tissues for energy utilization, lipid deposition, steroid hormone production, and bile acid formation. The lipoprotein consists of esterified and unesterified cholesterol, triglycerides, and phospholipids, and protein. The protein components of the lipoprotein are known as apolipoproteins (apo) or apoproteins. The different apolipoproteins serve as cofactors for enzymes and ligands for receptors. There are five major lipoproteins, each of which has a different function: Chylomicrons are very large particles that carry dietary lipid. They are associated with a variety of apolipoproteins, including A-I, A-II, A-IV, B-48, C-I, C-II, C-III, and E. Intermediate density lipoprotein (IDL) carries cholesterol esters and triglycerides. It is associated with apolipoproteins B-100, C-III, and E. Low density lipoprotein (LDL) carries cholesterol esters and is associated with apolipoprotein B-100. Very low density lipoprotein (VLDL) carries endogenous triglycerides and to a lesser degree cholesterol. The major apolipoproteins associated with VLDL are B-100, C-I, C-II, C-III, and E. High density lipoprotein (HDL) also carries cholesterol esters. It is associated with apolipoproteins A-I, A-II, C-I, C-II, C-III, D, and E. Understanding the major functions of the different apolipoproteins is important clinically, because defects in apolipoprotein metabolism lead to abnormalities in lipid handling. Apolipoproteins are usually measured by immunoassay or immunonephelometry. These techniques rely on measurement of the turbidity caused by apolipoprotein antibody complexes. A potential limitation of this method stems from the inherent turbidity of lipemic samples or even nonlipemic samples after repeated freezing and thawing. To some extent, automated systems can correct for such turbidity. Lipoprotein concentrations have been measured and described in several ways. Some of these measurements, including particle mass and mass concentration (the mass of each lipoprotein particle as solute per liter of solution), are not easily applied for screening or routine clinical purposes. Lipoprotein-associated phospholipase A2 (LP-PLA2) Cardiac Risk Assessment-Laboratory Tests Oct 15 20

Lipoprotein-associated phospholipase A2 (LP-PLA2) may play an important role in the pathophysiology of coronary heart disease (CHD). The polymorphism of LP-PLA2 gene caused LP-PLA2 enzyme activity depressing or lost. But there is not a definite conclusion for the association of between the LP-PLA2 gene polymorphism and CHD risk. To assess the relationship between LP-PLA2 gene V279F polymorphism and CHD, a comprehensive Meta-analysis was performed. All the case-control studies evaluating the association of between the LP-PLA2 gene V279F polymorphism and CHD risk were identified. Seven case-control studies involving 3,614 patients with CHD and 4,334 controls were included. The crude odds ratios (ORs) of meta-analysis under the different gene model were not significant. But in the stratified analysis by study size, ethnicity, cases definition, and source of controls under the additive model, the association was evident in ethnicity for Japanese group (OR=1.38, 95%CI=1.22-1.56), cases definition for MI (OR=1.22, 95%CI=1.01-1.49), source of controls for the based-hospital (OR=1.42, 95%CI=1.24-1.59). These data suggested that the V279F polymorphism in LP-PLA2 gene may contribute to CHD development. But there is necessary that more well-designed large studies are required for the validation of this association. Angiotensinogen Angiotensinogen is produced in the liver and cleaved by renin to make angiotensin I, which is cleaved by angiotensin converting enzyme (ACE) to make angiotensin II. There are two types of angiotensin II receptors, AT1 and AT2. AT1 has been better characterized and is thought to mediate ventricular and arterial hypertrophy. ACE inhibitors and angiotensin II receptor blockers are the pharmacologic agents used clinically to affect the renin-angiotensin system. Angiotensin II affects cardiac function indirectly (eg, via systemic arteriolar vasoconstriction and sodium and water retention) and directly via direct cardiac effects (eg, promotion of cardiomyopathy hypertrophy and maladaptive remodeling with LV dysfunction. Angiotensin gene (AGT) Plasma and tissue concentrations of ACE, and therefore levels of angiotensin II, are determined in part by the ACE gene, which is located on chromosome 17. This gene may manifest insertion (I) or deletion (D) polymorphisms and therefore three genotypes (DD, II, and ID). Plasma and cardiac levels of ACE are 1.5 to 3-fold higher in patients with the DD compared to those with II genotype; they are intermediate in patients with ID genotype. This is concordant with the observation that, in normotensive subjects, the acute effect of an ACE inhibitor is significantly greater and lasts longer in patients with the II ACE genotype. These data suggest that relatively low doses of ACE inhibitor may be sufficient for patients with low ACE activity and lower levels of angiotensin II while larger doses are required for those with high ACE activity. In addition, the DD genotype is associated with elevated levels of aldosterone, despite therapy with an ACE inhibitor, and an increased degradation of bradykinin. Hirschler et al. (2011) Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) has been proposed as a biomarker of risk of cardiovascular disease (CVD). To determine the association between Lp-PLA(2) activity and BMI, insulin-resistance, components of the metabolic syndrome (MS), and lifestyle behaviors in healthy adolescent boys. Data were collected cross-sectionally from 164 adolescents from an amateur rugby club. BMI, blood pressure (BP), Tanner stages, glucose, insulin, lipids, and Lp-PLA(2) Cardiac Risk Assessment-Laboratory Tests Oct 15 21

activity were measured. Questionnaires for lifestyle behaviors were completed. Approximately 26% of the adolescents were obese and 23% overweight. There was a univariate association between Lp-PLA(2) and BMI (r=0.16;p=0.042), triglycerides (r=0.26;p=0.001), LDL-C (r=0.46;p<0.001), apo B (r=0.55;p<0.001), whereas waist circumference, BP, glucose, HOMA-IR, and HDL-C were not correlated. None of the lifestyle behaviors were significantly correlated with Lp-PLA(2). In order to analyze Lp-PLA(2) association with known CVD risk conditions, adolescents were categorized according to overweight/obesity and to the presence of metabolic syndrome. Conversely, as it was for LDL-C and apo B concentration, Lp-PLA(2) activity was not higher in adolescents with obesity. Multiple regression analysis showed that apo B was significantly associated with Lp-PLA(2) adjusted for age, BMI, triglycerides and LDL-C (R2=0.32). Lp-PLA(2) activity was only associated with apo B adjusted for several confounding variables, suggesting that its clinical utility to identify individuals at risk for CVD does not surpass LDL-C and apo B in healthy adolescents. As plaque morphology may change with age, associations of Lp-PLA(2) with CVD may likewise vary with age. B-type Natriuretic Peptide (BNP) Testing B-type natriuretic peptide (BNP) is a cardiac neurohormone produced mainly in the left ventricle. It is secreted in response to ventricular volume expansion and pressure overload, conditions often present in congestive heart failure (CHF). Used in conjunction with other clinical information, measurement of BNP levels (either total or N-terminal) is useful in rapidly establishing or excluding the diagnosis of CHF in patients with acute dyspnea. Also, BNP levels determined in the first few days after an acute coronary syndrome or event may be useful in the prediction of longer-term cardiovascular risk. BNP measurements must be assessed in conjunction with standard diagnostic tests, medical history and clinical findings. The efficacy of BNP measurement as a standalone test has not been established yet. Moreover, certain conditions such as (and not limited to) ischemia, infarction and renal insufficiency, advanced age, female gender may cause elevation of circulating BNP; obesity, upstream heart failure and other conditions lower the BNP level. These conditions confound the interpretation of BNP levels to varying extents. The efficacy and/or utility of plasma BNP level as a monitor of the degree of CHF or the efficiency of CHF treatment has not been established. Treatment guided by BNP has not been shown to be superior to symptom-guided treatment in either clinical or quality of life outcomes. Therefore, BNP measurements for monitoring and management of CHF are non-covered. The efficacy but not the utility of BNP as a risk stratification tool (i.e., to assess risk of death, myocardial infarction or congestive heart failure) among patients with acute coronary syndrome (myocardial infarction with or without T-wave elevation and unstable angina) has been established. However, the assessment of BNP level has not been shown to alter patient management. The BNP is not sufficiently sensitive to either preclude or necessitate any other evaluation or treatment in this group of patients. ATP III ATP III recognizes additional positive risk factors for CHD, including elevations in Lp(a), remnant lipoproteins, small LDL particles, fibrinogen, homocysteine, highsensitivity C-reactive protein (hs-crp), impaired fasting plasma glucose (110 125 Cardiac Risk Assessment-Laboratory Tests Oct 15 22

mg/dl), and preexisting subclinical atherosclerosis (as evidenced by myocardial ischemia on exercise testing, carotid intimal medial thickening, and/or coronary artery calcium deposition). The links between some of these factors and CHD are obvious or are discussed elsewhere; however, others require expanded consideration. For example, elevated homocysteine levels have been linked to medications, as well as to genetic, disease and lifestyle conditions, and may contribute to CHD at least partially by exerting toxic effects on the endothelium. The links between hs-crp (a marker of chronic inflammation) and fibrinogen (a marker of prothrombotic states) and CHD at this point remain largely statistical; clear mechanisms have not been established. Elevation of apob (present in chylomicrons, VLDL, IDL, and LDL), decreases in apoa-i (present mostly in HDL), and increases in the apob/apoa-i ratio have also been associated with increased risk of CHD (Walldius, 2004). Although these risk factors are not used widely for screening purposes, they may be useful in defining risk status and refining treatment in patients already known to be at risk. Serum Uric Acid Neogi et al. (2011) The National Heart, Lung, and Blood Institute Family Heart Study is a multicenter study designed to assess risk factors for heart disease. Participants were recruited from population-based cohorts in the US states of Massachusetts, North Carolina, Minnesota, Utah, and Alabama. CAC was assessed with helical computed tomography (CT). The authors conducted sex-specific and family-cluster analyses, as well as additional analyses among persons without risk factors related to both cardiovascular disease and hyperuricemia, adjusting for potential confounders as we had in the previous study of carotid atherosclerosis. For the CAC study, 2412 subjects had both SUA and helical CT results available (55% women, age 58 ± 13 yrs, body mass index 27.6 ± 5.3). We found no association of SUA with CAC in men or women [OR in men: 1.0, 1.11, 0.86, 0.90; women: 1.0, 0.83, 1.00, 0.87 for increasing categories of SUA: < 5 (referent group), 5 to < 6, 6 to < 6.8, 6.8 mg/dl, respectively], nor in subgroup analyses. Replicating the methods used to demonstrate an association of SUA with carotid atherosclerosis did not reveal any association between SUA and CAC, suggesting that SUA likely does not contribute to atherosclerosis through effects on arterial calcification. The possibility that urate has divergent pathophysiologic effects on atherosclerosis and artery calcification merits further study. Fibrinogen Ferraro et al (2012) completed a study with the goal to assess the prognostic value of increased fibrinogen concentrations in ST-elevation myocardial infarction (STEMI) patients treated with primary percutaneous coronary intervention (PCI). A total of 428 STEMI patients treated with primary PCI were retrospectively selected (median age: 62 years; 82.5% males) from a continuous case series of 832 ACS patients. Plasma fibrinogen concentrations were measured before PCI and after 24, 48, and 72 hours. In the 4-year follow-up, one major adverse cardiovascular event (MACE) occurred in 111 patients (40%): 17 re-stemi (7%), 64 re-pci (22%), 22 cardiac deaths (7%), and eight non ST-elevated acute coronary syndromes (NSTEACS, 4%). According to the reference change value, fibrinogen concentrations increased in 25% of patients at 24 h, 64% at 48 h and 19% at 72 h. Only fibrinogen concentrations at 48 h showed a mild association with overall MACEs (p = 0.036): the risk increased, starting from a concentration of 4 g/l. However a further multivariate model did not confirm any prognostic value. No association with specific MACEs emerged. In contrast to NSTEACS patients, fibrinogen concentrations increased slightly in STEMI patients after primary PCI, however, they were not as prognostic as for MACEs. Cardiac Risk Assessment-Laboratory Tests Oct 15 23

Garg (2011) Predictive models for future risk of coronary heart disease (CHD) based on traditional risk factors, such as age, male gender, LDL cholesterol, HDL cholesterol, diabetes mellitus, hypertension, smoking and family history of premature CHD, are quite robust but leave room for further improvement. Thus, efforts are being made to assess additional biomarkers for CHD, such as, lipoprotein (a), C-reactive protein, fibrinogen, lipoprotein-associated phospholipase A2, homocysteine and others. However, none of the novel biomarkers has demonstrated improved prediction beyond traditional risk factor models in a consistent fashion across multiple cohorts. Many criteria have to be fulfilled before a biomarker can be considered clinically relevant. Another way is to develop new models predicting longterm or life-time risk of CHD. Further research using novel biomarkers and long-term predictive models has the potential to improve CHD risk prediction. The American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) have not updated their guidelines from 2010. Scientific Rationale Update July 2011 In December 2010, the American College of Cardiology Foundation and the American Heart Association Task Force (ACC/AHA) issued practice guidelines for assessment of cardiovascular risk in asymptomatic adults. The guideline notes that global risk scores (such as the Framingham Risk Score) that use multiple traditional cardiovascular risk factors should be obtained for risk assessment in all asymptomatic adults without a clinical history of coronary heart disease (CHD). They note these scores are useful for combining individual risk factor measurements into a single quantitative estimate of risk that can be used to target preventive interventions. The ACC/AHA guideline does not recommend measurement of lipid parameters, including lipoproteins, apolipoproteins, particle size, and density, beyond a standard fasting lipid profile for cardiovascular risk assessment in asymptomatic adults. In addition, measurement of natriuretic peptides is not recommended for CHD risk assessment in asymptomatic adults. The guideline states lipoprotein-associated phospholipase A2 (Lp-PLA2) might be reasonable for cardiovascular risk assessment in intermediate-risk asymptomatic adults, however, they note the usefulness/efficacy is less well established based on greater conflicting evidence from a single randomized trial or nonrandomized studies. According to the ACC/AHA guideline, measurement of CRP is not recommended for cardiovascular risk assessment in asymptomatic high-risk adults or low-risk men younger than 50 years of age or women 60 years of age or younger. The guideline states measurement of CRP can be useful in the selection of patients for statin therapy in men 50 years of age or older or women 60 years of age or older with lowdensity lipoprotein cholesterol less than 130 mg/dl; not on lipid-lowering, hormone replacement, or immunosuppressant therapy; without clinical CHD, diabetes, chronic kidney disease, severe inflammatory conditions, or contraindications to statins. This recommendation is a class IIa recommendation (i.e., recommendation in favor of treatment or procedure as useful/effective. Some conflicting evidence from single randomized trial or nonrandomized study.) They also note that measurement of CRP may be reasonable for cardiovascular risk assessment in asymptomatic intermediaterisk men 50 years of age or younger or women 60 years of age or younger. This recommendation is a class IIb recommendation (i.e., usefulness/efficacy less well Cardiac Risk Assessment-Laboratory Tests Oct 15 24

established. Greater conflicting evidence from single randomized trial or nonrandomized studies) Hatoum et al (2011) sought to determine the relation between and discriminative capability of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and coronary heart disease (CHD) in a large population of disease-free women. Among participants of the Nurses' Health Study who provided a blood sample, there were 421 cases of incident myocardial infarction during 14 years of follow-up. Controls were matched to cases 2:1 using risk set sampling based on age, smoking, and blood draw date. After conditioning on the matching factors, Lp-PLA(2) activity was significantly associated with myocardial infarction (relative risk [RR] 2.86 for extreme quartiles, 95% CI 1.98-4.12). Upon additional adjustment for lipid, inflammatory, and clinical risk factors, the RR remained statistically significant (RR 1.75, 95% CI 1.09-2.84). The discriminative capability of Lp-PLA(2) was assessed by comparing the area below the receiver operating characteristic curves for models with and without Lp-PLA(2) and by calculating the net reclassification improvement index. The addition of Lp- PLA(2) activity to a multivariable-adjusted model increased the receiver operating characteristic curves from 0.720 to 0.733 and significantly improved the net reclassification improvement index (P =.004). Investigators concluded levels of Lp- PLA(2) activity were significantly associated with incident CHD among women. In addition, Lp-PLA(2) activity added significantly to CHD risk discrimination. Jenny et al (2010) examined associations between lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) antigen level (mass) and enzymatic activity (activity) and cardiovascular disease (CVD) in older adults in a prospective observational study. Included in the study were patients randomly sampled from Medicare eligibility lists not adequately described. Associations of Lp-PLA(2) mass and activity with incident myocardial infarction (MI; n=508), stroke (n=565) and CVD death (n=665) using Cox regressions adjusted for age, sex, ethnicity and CVD risk factors in 3949 older adults, aged > 65 years at baseline, from the Cardiovascular Health Study (CHS) were examined. Lp-PLA(2) was reported to be associated with incident CVD events in these older adults. Hazard ratios for highest versus lowest tertiles of Lp-PLA(2) mass were 1.49 (1.19-1.85) for MI, 1.21 (0.98-1.49) for stroke and 1.11 (0.92-1.33) for CVD death. The highest tertile of Lp-PLA(2) activity was associated with MI (1.36; 1.09-1.70) and CVD death (1.23; 1.02-1.50). Combined Lp-PLA(2) tertile 3 and CRP>3mg/l, compared to Lp-PLA(2) tertile 1 and CRP<1mg/l, was associated with MI (2.29; 1.49-3.52) for Lp-PLA(2) mass and MI (1.66; 1.10-2.51) and CVD death (1.57; 1.08-2.26) for activity. For MI, both mass and activity added excess risk to elevated CRP alone (approximately 20% excess risk) and activity added excess risk for CVD death (approximately 12%). They concluded Lp-PLA(2) mass and activity were associated with incident CVD events in older adults in CHS. Lp-PLA(2) and CRP were independent and additive in prediction of events. While associations were modest, these results support further exploration of Lp-PLA(2) to identify older individuals at risk for CVD. Anuurad et al (2010) investigated the association between Lp-PLA(2) and CAD in a biethnic African-American and Caucasian population. Lp-PLA(2) mass, activity, and index, an integrated measure of mass and activity, and other cardiovascular risk factors were determined in 224 African-Americans and 336 Caucasians undergoing coronary angiography. The distribution of Lp-PLA(2) levels and determined the predictive role of Lp-PLA(2) as a risk factor for CAD were assessed. Levels of Lp- PLA(2) mass and activity were higher among Caucasians compared with African- Americans (293 +/- 75 vs. 232 +/- 76 ng/ml, P < 0.001 for mass and 173 +/- 41 vs. Cardiac Risk Assessment-Laboratory Tests Oct 15 25

141 +/- 39 nmol/min/ml, P < 0.001 for activity, respectively). However, Lp-PLA(2) index was similar in the two groups (0.61 +/- 0.17 vs. 0.64 +/- 0.19, P = NS). In both ethnic groups, Lp-PLA(2) activity and index was significantly higher among subjects with CAD. African-American subjects with CAD had significantly higher Lp- PLA(2) index than corresponding Caucasian subjects (0.69 +/- 0.20 vs. 0.63 +/- 0.18, P = 0.028). In multivariate regression analyses, after adjusting for other risk factors, Lp-PLA(2) index was independently (odds ratio 6.7, P = 0.047) associated with CAD in African-Americans but not Caucasians. The investigators concluded Lp- PLA(2) activity and index was associated with presence of CAD among African- Americans and Caucasians undergoing coronary angiography. The findings suggest an independent impact of vascular inflammation among African-Americans as contributory to CAD risk and underscore the importance of Lp-PLA(2) as a cardiovascular risk factor. The study was limited because patients were recruited from population scheduled for coronary angiography and were more likely to be at high risk as compared to healthier population. In addition there was manufacturer involvement during study execution Scientific Rationale Update October 2010 Diagnosis of obstructive coronary artery disease (CAD) in stable chest pain patients includes non-invasive testing, through functional and/or anatomical assessment of the heart and its vessels, including stress echocardiography, myocardial perfusion imaging and the gold standard of coronary angiography. Obstructive CAD is defined as at least one atherosclerotic plaque causing 50% luminal diameter stenosis in a major coronary artery ( 1.5 mm lumen diameter) as determined by invasive quantitative coronary angiography (QCA). Cardiovascular (CV) disease is the leading cause of death and disability in the United States and has grown to become the leading health issue globally. Despite significant strides in secondary prevention and treatment, individualized risk assessment and primary prevention of coronary artery disease (CAD) remain major challenges. The majority of first myocardial infarctions occur in subjects without a known history of CAD and in whom proven preventive therapies would not have been indicated, according to current guidelines based on the Framingham Risk Score (FRS), prior to the event. The FRS performs reasonably well at a population level in differentiating groups at differing risk, but its deficiencies at the level of individual risk assessment have been increasingly recognized. Thus, a major goal of contemporary CV medicine is to discover and validate additional markers of CAD risk that might identify individuals with pre-clinical disease or assess the likelihood of CAD in patients who might be candidates for intervention. Three specific areas that promise to increase early detection of disease or improve on discovery of disease predisposition are (1) biomarkers, (2) vascular imaging, and (3) genomics. CardioDx has developed a gene expression-based blood test, called Corus CAD, that measures the expression levels, or activity, of 23 genes that quantifies the likelihood of obstructive coronary artery disease (CAD) in patients with stable chest pain. An algorithm is applied to the gene expression results to calculate a score that indicates the likelihood of the presence of obstructive coronary artery disease (CAD) in a patient. The score ranges from 0-40. Corus CAD is being proposed as of particular benefit to women because imaging modalities and chest pain type were less effective in the PREDICT trial, noted below, in terms of identifying patients who had obstructive CAD after being sent for an angiography. Women under 60 years of age are usually under low risk for coronary Cardiac Risk Assessment-Laboratory Tests Oct 15 26

disease, primarily because estrogen is cardioprotective and therefore have a different biological profile than men. There is currently a Clinical Trial "Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS)", sponsored by CardioDX, which is recruiting participants. The purpose of this study is to validate the use of Corus CAD blood assay in subjects who are referred for the work-up of coronary artery disease. The study will evaluate the clinical utility of a gene expression test (Corus CAD) in subjects referred for myocardial perfusion imaging (MPI) work-up for suspected obstructive atherosclerotic coronary artery disease (CAD). The Corus CAD is a gene expression test that is proposed to quantify the expression of multiple genes from circulating peripheral blood cells to detect the presence of clinically significant obstructive CAD in patients with chest pain. The study will enroll a patient population that presents with stable chest pain syndrome or anginal equivalent and referred for stress myocardial profusion imaging. The Estimated Study Completion Date is May 2011. ClinicalTrials.gov Identifier is NCT01117506. A reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR), a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification". RT-PCR based 23 gene expression test, Corus CAD, has been developed and validated for the assessment of obstructive CAD in the PREDICT multi-center trial in which all patients were referred to invasive coronary angiography. An analysis of gender specific performance of myocardial perfusion imaging (MPI) with Corus CAD in the PREDICT trial has not previously been reported. MPIs were performed during the clinical workup for the angiographic population in the PREDICT multi-center clinical validation study. MPIs were defined as positive if at least one reversible or fixed defect consistent with obstructive CAD was observed; indeterminate or intermediate defects were considered negative. Obstructive CAD was defined as 1 or more lesions with 50% stenosis by quantitative coronary angiography (QCA). In the PREDICT validation cohort (N=526, 57% men, 37% obstructive CAD) 310 patients had MPIs of which 223 were positive. In men 41% of the positive MPIs (58/142) had obstructive CAD by QCA, but only 23% (18/81) of women. The discordance between MPI and QCA may be partially due to clinicallydriven referral bias. Overall, the sensitivity and specificity of MPI were 78 and 31%, respectively, whereas for Corus CAD they were 87% and 40%. The Corus CAD score improved MPI based classification of obstructive disease patients by either reclassification or ROC analyses (p=2x10-8 and 2x10-6, respectively). For female MPI positive patients, 56% were reclassified correctly as low risk of obstructive CAD by the Corus CAD score. In the PREDICT study, positive MPI results were not significantly predictive of obstructive CAD, with decreased performance in women. The Corus CAD gene expression score added diagnostic value, improved MPI performance overall, and may be particularly useful in identifying false positive MPIs in women. However, additional, larger, peer-reviewed, randomized, controlled or comparable studies in a non-angiography referred population, will help determine the generality of these results. Corus CAD is now available in nine states, Kentucky, Maryland, Illinois, Washington, Wisconsin, Minnesota, North Carolina, Texas and Arizona, via the CardioDx CLIAcertified Commercial Laboratory. It is offered as a test in the physician office setting and is not recommended for patients with acute chest pain. Cardiac Risk Assessment-Laboratory Tests Oct 15 27

In summary, the majority of information on this topic is on websites sponsored by Cardio DX. Other than the PREDICT study noted above and the Clinical Trial, "Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS)" sponsored by Cardio DX, with the estimated completion date of May 2011, there are few peer-reviewed studies in the medical literature regarding this testing. Data from large-scale, well-designed, controlled or comparison trials are needed to establish the best patient criteria and demonstrate its effectiveness in the long-term. At this time, because of the paucity of studies, this would be considered investigational. Scientific Rationale Update March 2010 Coronary heart disease affects approximately 6% of all American adults and is the leading cause of death in the United States. This disease is caused by a decrease in blood flow to the heart due to atherosclerotic blockage of the coronary arteries, which results in decreased oxygen and nutrient delivery to the affected heart muscle. Lp-PLA2 is an enzyme in the blood that associates primarily with low-density lipoprotein (LDL). LDL carries Lp-PLA2 to the walls of arteries, where it can activate an inflammatory response, promoting atherosclerosis. Lp-PLA2 thus serves as a specific indicator of vascular inflammation, and the test attempts to identify individuals who are at high risk for coronary heart disease based on elevated Lp- PLA2 levels. Ischemic stroke occurs when a clot blocks an artery that supplies blood to the brain. Strokes such as this can impair brain function, resulting in disability or death. According to American Heart Association statistics, the annual incidence of stroke in the United States is 795,000 and the total number of people who have survived strokes is estimated to be 6.5 million. Almost 70% of attacks occur in persons aged 65 years and older and ischemic stroke accounts for approximately 88% of strokes in the United States. Tools for assessment of stroke risk have been developed and used to select patients for preventative treatments; however, a joint committee of the American Heart Association and American Stroke Association has stated that more research is needed to validate existing risk assessment tools and to determine whether recently identified risk factors can improve the accuracy of these tools. One such recently identified risk factor is elevated levels of lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme that can activate inflammation and promote narrowing and hardening of the arteries. The diadexus PLAC test is a blood test that can be ordered by a physician for the measurement of Lp-PLA2. According to the test manufacturer, it is indicated for use in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for ischemic stroke and coronary heart disease. The PLAC test uses antibodies that bind to Lp-PLA2 and reagents, which cause color formation that is proportional to the number of molecules of Lp-PLA2 in the blood or serum sample. Current assessment of stroke risk usually relies on tools that evaluate numerous clinical parameters known to be predictive of cardiovascular health. The Framingham Stroke Profile, the most widely used tool, calculates a gender-specific 10-year cumulative stroke risk using independent predictors such as age, systolic blood pressure, hypertension, diabetes mellitus, current smoking habits, established cardiovascular disease, atrial fibrillation, and left ventricular (LV) hypertrophy on electrocardiogram.8 The Framingham Stroke Profile has been updated recently to account for the use of antihypertensive therapy and the risks associated with newonset atrial fibrillation Cardiac Risk Assessment-Laboratory Tests Oct 15 28

Studies Although most of these studies involved more than 1000 patients, none of the studies examined the influence of treatment on patients considered to be at elevated risk of stroke and all of the studies were supported in some way by the test manufacturer. Persson et al. (2008) completed the largest study of the PLAC test for ischemic stroke, which enrolled 6103 adults aged 45 to 69 years who volunteered for a prospective evaluation of the influence of diet on cancer. This study was extended to an assessment of cardiovascular disease in a subgroup that consisted of 5250 adults (41% men, 59% women; mean age 57 years; mean body mass index [BMI] 26 kg/m2) who underwent the PLAC test and assessment of blood pressure, HDL, LDL, triglycerides, hs-crp, smoking status, alcohol consumption, presence of diabetes, and use of hormone replacement therapy (HRT). At a mean of 10.6 ± 1.7 years after baseline assessment, 152 (3%) patients had experienced an ischemic stroke and 12 (8%) of these strokes were fatal. Patients who experienced strokes were divided into Low-, Medium-, and High-level Groups based on mass of Lp-PLA2 detected by the PLAC assay. After Cox regression analysis to adjust results for differences in 11 variables including age, sex, HDL, LDL, BMI, hs-crp, diabetes, smoking, and systolic blood pressure, there was a statistically significant difference between Low-level and High-level Groups, which corresponded to a 1.9-fold increase in risk of first ischemic stroke for the High-level Group (P<0.05). A statistically significant 1.7-fold increase in risk of first stroke was also seen for the Medium-level versus Low-level Group (P<0.05). In addition, the trend of increasing risk with increasing Lp-PLA2 level was statistically significant after the same multivariable adjustment (P=0.008). Similar analysis indicated that there was not a statistically significant correlation between Lp-PLA2 level and coronary heart disease events in this study population. Sabatine et al. (2007) conducted a study that evaluated the PLAC test in 3766 patients (81% men, 19% women; mean age 64 years; mean BMI 29 kg/m2) who had stable coronary artery disease. Other parameters measured prospectively included hypertension, diabetes, smoking, prior myocardial infarction, prior coronary intervention, cholesterol level, hs-crp, glomerular filtration rate, and use of betablocker or lipid-lowering therapy. After a median of 4.8 years, 87 (2%) patients had nonfatal stroke. To evaluate the association between stroke and PLAC test results, patients were divided into 4 groups based on Lp-PLA2 levels and hazard ratios were calculated after multivariable Cox regression to adjust for differences in baseline variables and use of the antihypertensive trandolapril. Although there was a superficial association between increased Lp-PLA2 levels and increased risk of stroke, this association was not statistically significant (P=0.13 for the trend). In contrast, a statistically significant association was observed between increasing Lp-PLA2 levels and risk of acute coronary syndromes (P=0.002). In a study that evaluated the PLAC test for prediction of stroke risk in women, Wassertheil-Smoller et al. (2008) evaluated data for a subgroup of 1860 postmenopausal women (median age range 60-69 years, median BMI range 25-30 kg/m2; 39% undergoing HRT) in the Hormones and Biomarkers Predicting Stroke Study. 929 women who had an ischemic stroke were retrospectively matched with 929 control women based on age, ethnicity, date of study enrollment, and duration of follow-up. Patients were divided into 4 groups based on Lp-PLA2 levels and stroke risk adjusted for age, ethnicity, BMI, HDL, LDL, diabetes, smoking, systolic blood pressure, and use of aspirin and hypertensive medication. After these adjustments, Cardiac Risk Assessment-Laboratory Tests Oct 15 29

there was a statistically significant association between increasing Lp-PLA2 and risk of stroke in nonusers of HRT (P<0.01 for the trend). For nonusers of HRT, the quartile of patients with the highest Lp-PLA2 levels had a 1.5-fold increase in stroke risk compared with the quartile of patients with the lowest Lp-PLA2 levels (P<0.05). The latter association includes an adjustment for differences in CRP levels. Furthermore, there was a statistically significant 2.3-fold increase in risk of stroke for nonusers of HRT who had high versus low levels of Lp-PLA2 and CRP. In contrast with these observations for nonusers of HRT, there was no statistically significant association between stroke risk and Lp-PLA2 levels for users of HRT or for all patients combined. A large retrospective study that evaluated the PLAC test was performed by Ballantyne et al. (2005) and compared Lp-PLA2 levels and other risk factors for 194 adults who had an ischemic stroke (mean age 60 years, 44% women, 34% current smokers) versus 766 adults who did not have a stroke (mean age 57 years, 58% women, 24% current smokers). There was no significant increase in stroke risk for the moderate Lp-PLA2 level subgroup compared with the lowest level subgroup. Essentially the same results were obtained when BMI and use of antihypertensive medication were included in the risk estimation. In a second article by the same group of investigators, the data obtained above were reanalyzed to assess overall diagnostic accuracy of the PLAC test for prediction of ischemic stroke based on areaunder-the-curve analysis. Addition of PLAC test data resulted in a statistically significant 3% to 4% increase in area-under-the-curve compared with traditional risk factors alone or traditional risk factors combined with hs-crp (P<0.05). Elkind et al. (2006) performed a prospective study that evaluated the PLAC test for assessment of stroke risk and that enrolled 467 patients (mean age 69 years, 45% men) who had experienced a first ischemic stroke. For 420 (90%) patients, stroke occurred < 7 days before enrollment in the study. Blood tests including the PLAC test were performed at the time of hospital admission for stroke. During a median of 4 years follow-up, stroke recurred in 80 (17%) patients and 15 (3%) patients died as a result of a recurrent stroke; however, the mean time to stroke recurrence was not reported and patients were not excluded for stroke recurrence shortly after initial stroke. To estimate the risk of recurrent stroke associated with Lp-PLA2, patients who did and did not have recurrent stroke were divided into 4 subgroups based on their Lp-PLA2 levels and adjustments were made for differences in age, sex, ethnicity, coronary artery disease history, diabetes, hypertension, hyperlipidemia, atrial fibrillation, smoking status, and hs-crp levels. After factoring in these adjustments, the subgroup with the highest Lp-PLA2 levels had a 2.08-fold (95% CI 1.04-4.18) increase in the risk of stroke recurrence and a 1.86-fold (95% CI 1.01-3.42) increase in risk of myocardial infarction, recurrent stroke, and death from vascular events combined. Subgroup analysis indicated that increased Lp-PLA2 levels were not associated with a statistically significant increase in death from all causes. The peer-reviewed literature search did not identify studies, which demonstrated that accepted available treatments can reduce the risk of stroke in patients who have elevated Lp-PLA2. Consequently, the PLAC test may provide little clinically useful information, since there may not be any effective therapies to reduce stroke risk in patients who have significantly elevated levels of Lp-PLA2. Further studies are needed to determine whether the PLAC test provides clinically relevant diagnostic information. Cardiac Risk Assessment-Laboratory Tests Oct 15 30

Scientific Rationale Update July 2009 The VAP (Vertical Auto Profile) Cholesterol Test, was developed by Atherotech, Inc., a cardio-diagnostic company, in 2006. VAP also refers to vertical density gradient ultracentrifugation. This is a measure of lipid testing, that is being advertised as the first cholesterol profile to comply with updated recommendations calling for more accurate, direct low-density lipoprotein (LDL) measurement unaffected by triglycerides. It is also the only commercially available advanced lipid profile that routinely reports all three lipoprotein parameters considered necessary by the American Diabetes Association and American College of Cardiology expert consensus guidelines. The VAP Cholesterol Test is available through national and regional diagnostic laboratories. The majority of the information on VAP was primarily submitted from the developer or those associated with the product. The Atherotech VAP Method is noted below: Fractionation based on density gradient ultracentrifugation Rapid separation on short vertical axis, re-orients Cholesterol analysis of effluent from tubes provides particle density profile Overlapping subclasses quantitation depends on proprietary software for deconvolution of profile using algorithm based on purified UC fractions Provides a size phenotype, A, B or AB Measures Lp (a) cholesterol content Provides non-hdl cholesterol Separate tests for TG, hscrpand homocysteine With the VAP method, the major lipoprotein groups can be sorted by density into subcomponents, including four levels of LDL and at least three levels of each of the other major lipoprotein groups. The VAP evaluation includes measures of non-hdl cholesterol, total LDL cholesterol, and its three components: lipoprotein (a) cholesterol, IDL cholesterol, and real LDL cholesterol. Ensign et al. (2006) completed a study to clarify the differences in analytical results, from a clinical perspective, among four technologies currently used in clinical reference laboratories for the analysis of LDL subfractions: gradient gel electrophoresis (GGE), ultracentrifugation-vertical auto profile (VAP), nuclear magnetic resonance (NMR), and tube gel electrophoresis (TGE). Four simultaneous blood samples were collected from 40 persons (30 males and 10 females) to determine LDL subclasses in four different clinical reference laboratories using different methods for analysis. LDL subfractions were assessed according to LDL particle size, the results categorized according to LDL phenotype, and the heterogeneity of results were observed among the 4 methods. Complete agreement among methods with respect to LDL subclass phenotyping occurred in only 8% (n = 3) of the persons studied. NMR and GGE agreed most frequently at 70% (n = 28), whereas VAP matched least often. As measurement of LDL subclasses becomes increasingly important, standardization of methods is needed. Variation among currently available methods renders them unreliable and limits their clinical usefulness. At this time, there is inadequate evidence in the peer-reviewed medical literature to support the efficacy of the VAP (Vertical Auto Profile) Cholesterol Test. There are no available RCT that compare VAP to the current testing used for cholesterol to determine if the outcomes are equal or better than testing currently being used. The study by Ensign et al. (2006) notes that variation among currently available methods Cardiac Risk Assessment-Laboratory Tests Oct 15 31

renders them unreliable and limits their clinical usefulness. As noted in this email, the majority of the literature on VAP was by the developers of the test or those with interest in the product. In addition, because of the paucity of the reviews on VAP, the long-term safety and efficacy has not been determined. VAP is therefore considered investigational at this time. Scientific Rationale Update February 2009 Inflammation plays a critical role in the development of vascular disease. Increased levels of the inflammatory biomarkers, lipoprotein-associated phospholipase A2 (Lp- PLA2), and high-sensitivity C-reactive protein (hs-crp) may be associated with an increased risk for ischemic stroke. Measuring C-reactive protein (CRP) has also been recommended to identify patients at high risk for coronary heart disease (CHD) with low LDL cholesterol (LDL-C). However, the key outcome of cardiac risk assessment is an improvement in health outcomes. Improved risk prediction does not by itself result in improved health outcomes. To improve outcomes, clinicians must have the tools to translate this information into clinical practice. To do this requires guidelines that incorporate emerging risk factors into existing risk prediction models and that have been demonstrated to classify patients into risk categories with greater accuracy. There have been a number of studies related to lipoprotein-associated phospholipase A2 (Lp-PLA2) and high-sensitivity C-reactive protein (hs-crp) testing for prediction of coronary heart disease and ischemic stroke. The original ARIC study was a prospective randomized trial initiated between 1987 and 1989 in four US communities. This study assessed the association between the metabolic syndrome and long-term CVD morbidity and mortality in the biracial population. A nested case cohort study took this completed large RCT, (in this situation, it was the 2009 ARIC Trial), and reanalyzed the data according to a specific protocol. In this study, everyone who had the outcome of a stroke was compared to a control group who did not have a stroke from the same trial. The stroke patients were (n=949) out of 12,762 apparently healthy, middle-aged men and women were examined to determine whether Lp-PLA2 and hs-crp levels improved, in the area under the curve (AUC), for a 5-year ischemic stroke risk. The AUC is a way to combine sensitivity and specificity into one measure. The authors examined how Lp- PLA2 and hs-crp levels altered classification of individuals into low, intermediate, or high-risk categories compared with traditional risk factors. The authors conclude that Lp-PLA2 and hs-crp may be useful in individuals classified as intermediate risk for ischemic stroke by traditional risk factors. However, there is no evidence-based determination that Lp-PLA2 and hs-crp would change the likelihood of stroke. Ballantyne et al. (2005) and Elkind et al. (2006) found that the subgroup of patients with the highest levels of Lp-PLA2 had a statistically significant 2.0- to 2.1-fold increase in risk of stroke or recurrent stroke compared with the subgroup of patients with the lowest levels of Lp-PLA2. However, the reviewed studies overestimated the magnitude of the predictive ability of the PLAC test by subdividing the study populations into 3 or 4 subgroups and comparing only those patients who have the highest and lowest levels of Lp-PLA2. Several research groups have joined to form the Lp-PLA2 Studies Collaboration, which is compiling data from their observational epidemiological studies. By combining this data with information about factors affecting Lp-PLA2 measurements, Cardiac Risk Assessment-Laboratory Tests Oct 15 32

the investigators hope to obtain sufficient data to enable accurate Lp-PLA2 analysis in subgroups that differ in their ages, genders, smoking status, and other characteristics. When completed, the database will provide information concerning approximately 15,000 cardiovascular disease endpoints. In addition, the available studies have not determined whether information provided by PLAC testing can be used effectively to guide treatment and improve patient outcomes. The results of these studies fail to provide conclusive evidence that the PLAC test can improve stroke prediction of management of patients who may be at increased risk for stroke. Although Lp-PLA2 has been classified as a factor that may be indicative of stroke risk, the guidelines cautioned that no studies have been performed to determine whether reduction in Lp-PLA2 level provides clinical benefits such as stroke prevention. Demand for the PLAC test may increase if further studies indicate that this testing improves the accuracy of stroke risk prediction. Additional, randomized controlled trials are necessary to establish whether therapies that lower LpPLA2 reduce cardiovascular risk. Scientific Rationale Update February 2008 According to the National Cholesterol Education Program Adult Treatment Panel (ATPIII) lipoprotein particles or remnants, including intermediate density lipoproteins (IDLs) and VLDLs have been shown to be atherogenic. They are triglyceride-rich lipoproteins, and elevated triglycerides have been identified as an independent risk factor of CVD. The lipoprotein remnant particles may penetrate the arterial wall more easily than larger lipoproteins. The panel concluded that studies are limited, and measurement with specific assays for lipoprotein remnants cannot be recommended for routine practice. According to a statement from the American Heart Association (AHA) and the American College of Cardiology (ACC) regarding the use of conditional and predisposing risk factors in risk assessment, conditional risk factors include: elevated serum triglycerides, small LDL particles, elevated serum homocysteine, elevated serum lipoprotein (a), prothrombotic factors (e.g., fibrinogen), and inflammatory markers (e.g., C-reactive protein). However, their quantitative contribution and independence of contribution to risk are not well defined, and they are not usually included in the global risk assessment. In addition, the AHA and ACC concluded a high serum concentration of homocysteine is associated with increased risk for CHD; however, it remains to be proven in controlled clinical trials that a reduction in serum homocysteine levels will reduce the risk for CHD. Routine measures of lipoprotein(a), fibrinogen, and C-reactive protein currently are not recommended. An elevated serum lipoprotein(a) correlates with a higher incidence of CHD in some studies but not in others, and specific therapeutics to reduce lipoprotein(a) levels is not available. Additionally, the AHA and ACC stated that some investigators have suggested that an elevated lipoprotein(a) level justifies a more aggressive lowering of LDL C. Furthermore, an elevated fibrinogen level is also correlated with a higher CHD incidence; however, again, no specific therapies are available, except that in smokers, smoking cessation may reduce fibrinogen concentrations. Finally, C-reactive protein is promising as a risk predictor. The preferred method for measurement appears to be a high-sensitivity test. C-reactive protein appears to be related to systemic inflammation; however, its causative role in atherogenesis is uncertain. LDL particle size can be measured by several techniques, including ultracentrifugation, gradient gel electrophoresis and nuclear magnetic resonance spectroscopy (NMR LipoProfile). Ultracentrifugation measures Cardiac Risk Assessment-Laboratory Tests Oct 15 33

the flotation velocity of LDL and is the gold standard, although it is only available in a few research laboratories; gradient gel electrophoresis specifies the average size of the LDL particle and is more widely available. NMR measures the diameter and lipid concentration of LDL (Sacks and Campos, 2003). Manufacturers of NMR LipoProfile (LipoScience Inc., Raleigh, NC) have recommended testing as a method of observing subtle changes in cholesterol particles resulting from specific interventions, allowing for maximized risk reduction, particularly for patients that are at high risk for heart disease and/or metabolic syndrome. No standards for LDL subclass categorization and optimal levels of the LDL subclasses have been established. Due to the lack of consensus regarding optimal patient-selection criteria, standardization of levels and insufficient supporting evidence, it is unknown what role LDL subclass testing has in the routine screening, diagnosis or patient management for the treatment of CVD. Other risk factors such as apolipoproteins, angiotensinogen gene testing (e.g., CardiaRisk), lipoprotein remnants, LDL and HDL subclasses, Lp PLA2 and prothrombotic factors, in addition to long-chain omega-3 fatty acid testing and interleuking6 174 polymorphism, have not been proven to be independent risk factors, and routine testing is not supported at this time. Furthermore, it has not been shown that reducing levels have improved patient outcomes. Further clinical trials are needed. Although some studies found NMR LipoProfile analysis of the total number of LDL particles or the number of small LDL particles positive independent predictors of CHD in primary prevention, a recent large study did not find that NMR analysis was an independent factor in the prediction of CHD risk for primary prevention. The data supporting NMR analysis as an independent risk factor for secondary prevention of CHD are more limited and more studies are needed before NMR analysis of lipoproteins can become an accepted risk factor. Further studies are needed especially to investigate the clinical consequences of the inadequacies of the standard lipid panel. Controlled studies are needed that compare groups of patients using the NMR test with those using standard lipid panels for primary and secondary prevention of CHD and also for physicians seeking to optimize patients ongoing treatment. Scientific Rationale Update March 2007 Accumulating evidence has suggested that Lp-PLA2 is a biomarker of coronary artery disease and may have a proinflammatory role in the progression of atherosclerosis. The recognition that atherosclerosis represents, in part, an inflammatory process, has created considerable interest in measurement of proinflammatory factors as part of cardiovascular disease risk assessment. Recently, the FDA cleared for marketing an enzyme linked immunoabsorbent assay (ELISA) test; PLAC test. Results of two large-scale observational studies have suggested that Lp-PLA2 is an independent risk factor for coronary heart disease in men. For example, Packard et al (2000) published the results of their West of Scotland Coronary Prevention Study (WOSCOPS) which was a five-year case control trial evaluating 6,595 men with elevated cholesterol and no history of a heart attack. Researchers looked at a smaller population of this study group to determine if inflammatory markers such as Lp-PLA2 and high sensitivity C-reactive protein were correlated with coronary heart disease events. The 580 men who went on to have a myocardial infarction or revascularization were compared to 1,160 age and smoking matched men who did Cardiac Risk Assessment-Laboratory Tests Oct 15 34

not have an event. The results showed that those with the highest levels of Lp-PLA2 had twice the risk of an event, compared to those with the lowest levels, even after adjustment for traditional risk factors and other inflammatory mediators. Ballantyne et al (2002) evaluated various risk markers and their association with increased risk in over 12,000 individuals in The Atherosclerosis Risk in Communities (ARIC) study. At enrollment in the study, patients were free of coronary heart disease and followed for the development of the disease for the next nine years. The case-cohort component of the study examined two inflammatory markers, Lp-PLA2 and high sensitivity C-reactive protein in a subset of 609 cases and 741 controls. The results showed that levels of Lp-PLA2 are higher in those who developed coronary heart disease cases. In individuals with non-elevated LDL levels (<130 mg/dl), Lp- PLA2 levels were independently associated with coronary heart disease, even after adjustment for traditional risk factors and C-reactive protein. As noted in the FDA press release accompanying the FDA approval for the PLAC test, An elevated PLAC test result with an LDL-cholesterol level of less than 130 mg/dl gives doctors increased confidence that patients have two to three times the risk of having coronary heart disease when compared with patients having lower PLAC test results. The MONICA Study, a German sub study of the Monitoring Trends and Determinants in Cardiovascular Diseases (MONICA) was an international collaborative study with the objective of measuring over 10 years, and in many different populations, the trends in, and determinants of, cardiovascular disease. The study evaluated the relationship between Lp-PLA2 levels and risk of coronary events. A total of 934 apparently healthy men were followed for 14 years, with 97 men experiencing a coronary event. The study revealed that elevated levels of Lp-PLA2 were predictive of future coronary events in apparently healthy middle-aged men with only moderately elevated total cholesterol. Elevated Lp-PLA2 levels did not correlate with CRP, and were shown to be additive in their ability to predict risk of coronary heart disease. In the Mayo Heart Study, 504 consecutive patients undergoing clinically indicated coronary angiography were monitored for major adverse events during a four-year follow-up. Seventy major adverse events occurred in 61 of 466 (13%) contacted patients (20 deaths, 14 myocardial infarctions, 28 coronary revascularizations, and 10 strokes). The study evaluated the association of Lp-PLA2 with coronary artery disease (CAD) risk factors, with the severity of angiographic CAD, and with the incidence of major adverse events. The study found that higher Lp-PLA2 levels were associated with a significantly greater risk of events (death, MI, revascularization, stroke). The risk of an event increased by 28% with each standard deviation increase in Lp-PLA2. The association remained significant after adjusting for clinical variables (age, gender, smoking, hypertension) and lipids (total and HDL cholesterol, Lp(a), and triglycerides) as well as CRP and fibrinogen. Mitchell et al (2006) conducted a population-based study of stroke risk factors in 467 patients with first ischemic stroke to determine whether levels of high-sensitivity C- reactive protein (hs-crp) and lipoprotein-associated phospholipase A2 (Lp-PLA2) predict risk of stroke recurrence, other vascular events, and death. They found that levels of Lp-PLA2 and hs-crp were weakly correlated. High-sensitivity CRP, but not Lp-PLA2, was associated with stroke severity. After adjusting for age, sex, race and ethnicity, history of coronary artery disease, diabetes mellitus, hypertension, hyperlipidemia, atrial fibrillation, smoking, and hs-crp level, compared with the lowest quartile of Lp-PLA2; those in the highest quartile had an increased risk of Cardiac Risk Assessment-Laboratory Tests Oct 15 35

recurrent stroke and of the combined outcome of recurrent stroke, MI, or vascular death. After adjusting for confounders, hs-crp was not associated with risk of recurrent stroke or recurrent stroke, myocardial infarction, or vascular death but was associated with risk of death. The authors concluded that inflammatory markers are associated with prognosis after first ischemic stroke and may offer complementary information. Lipoprotein-associated phospholipase A2 may be a stronger predictor of recurrent stroke risk. Levels of hs-crp, an acute-phase reactant, increase with stroke severity and may be associated with mortality to a greater degree than recurrence. Despite the above findings, the sine qua non of cardiac risk assessment is an improvement in health outcomes. Improved risk prediction does not, by itself, result in improved health outcomes. To improve outcomes, clinicians must have the tools to translate this information into clinical practice. This requires guidelines that incorporate emerging risk factors into existing risk prediction models and that have been demonstrated to classify patients into risk categories with greater accuracy. Predictive models also need to be accompanied by treatment guidelines that target intervention toward patients who will get the most benefit. At the present time, measurements of Lp-PLA2 are not a component of the guidelines developed by the National Cholesterol Education Program Adult Treatment Panel III. While studies have suggested that statin drugs and fibrates may reduce levels of Lp-PLA2, it is not known whether such drug therapy in patients not already considered candidates, based on other well established risk factors, will ultimately decrease the incidence of coronary heart disease. Moreover, a very recent study by Folsom and colleagues (2006) investigated the role of 19 novel risk markers in the risk assessment for coronary artery disease. This study included 15,792 participants initially evaluated between 1987 and 1989 that were followed to 2005. The results of the study indicate that the addition of novel risk markers, including Lp-PLA2, to standard risk assessment factors (age, race, gender, total and HDL cholesterol, systolic blood pressure, hypertensive medication use, smoking status and the presence of diabetes) does not significantly improve the predictive power of coronary risk screening. In an accompanying editorial, Lloyd- Jones and Tian (2006) state, the ultimate measure of a novel screening test is the ability reclassify individuals. In other words, a new marker is useful only when it corrects a substantial portion of misclassification by the old test. Using that as the measure of utility, the results of the Folsom study lead to the conclusion that the use of novel risk markers is not warranted. Scientific Rationale - Initial Some 7 million Americans suffer from coronary heart disease (CHD), the most common form of heart disease. This type of heart disease is caused by a narrowing of the coronary arteries that feed the heart. Each year, more than 500,000 Americans die of myocardial infarctions caused by CHD and this represents the number one killer of both men and women in the United States. In fact, the number of deaths attributed to cardiovascular disease is greater than the number resulting from all other leading causes of death combined, including cancer, accidents, and HIV infection. Therefore, it is imperative to understand not only how to diagnose heart disease but also how to stratify its risk, an important factor in treatment decision making in patients with coronary artery disease. Once the initial diagnosis has been made, various cardiac tests can be used to obtain pertinent information about risk level and the appropriateness of medical versus more invasive treatments. Cardiac Risk Assessment-Laboratory Tests Oct 15 36

Recent studies have suggested that low level chronic inflammation may play a role in atherogenesis, and thus measurement of C-reactive protein (CRP) has been investigated for risk assessment in primary prevention of cardiovascular diseases. While a regular CRP test is adequate for evaluation of a clinically significant inflammatory process, it lacks the sensitivity needed for risk assessment of cardiovascular diseases. High-sensitivity C-reactive protein (hs-crp) is a relatively new test that has been widely publicized as a means to evaluate a patient's risk of heart attack or other heart conditions. The hs-crp test differs from the CRP test in its ability to detect small amounts of CRP in the blood; hence the term "highsensitivity" CRP. Studies suggest that hs-crp is useful in detecting the small amounts of CRP in patients with atherosclerosis, an inflammatory disease that reduces the flow of blood to the heart and puts the patient at risk of a heart attack. Current data suggest that the addition of hs-crp to standard lipid screening can improve the ability to detect absolute coronary risk. This is a critical issue because one-half of all myocardial infarctions and strokes occur among individuals without overt hyperlipidemia. An expert panel convened by the American Heart Association (AHA) and the Centers for Disease Control (CDC) has confirmed the benefits of hs-crp for some patients at risk of a heart attack but recommends against its use as a screen for the general population. The panel's conclusions confirm that hs-crp may be of value to physicians who are deciding on treatment options for patients who have a 10 to 20% risk of heart attack in the next 10 years, as determined by global risk assessment using Framingham point scoring. In such situations, the hs-crp test can help determine whether the physician recommends an aggressive treatment or something more moderate. The report further points out that no evidence currently suggests that hs-crp testing in high-risk patients has an impact on their treatment outcomes, and so, testing in high-risk patients is not recommended. On July 21, 2003, The Food and Drug Administration (FDA) cleared for marketing the PLAC Test, an enzyme immunoassay for the quantitative determination of lipoprotein-associated phospholipase A2 (Lp-PLA2) in plasma. Lp-PLA2 is an enzyme made by macrophages that can hydrolyze oxidized phospholipids to generate lysophosphatidylcholine and oxidized fatty acids, which have proinflammatory properties. The recognition that atherosclerosis represents, in part, an inflammatory process has created considerable interest in measurement of proinflammatory factors, as part of cardiovascular disease risk assessment. Some large-scale observational studies have suggested that Lp-PLA2 portends greatest increased risk in subjects with the highest PLAC test results and LDL cholesterol levels lower than 130mg/dL, while others found that prognosis based on Lp-PLA2 was not statistically significant after adjustment for other risk factors. There still is a lack of evidence from prospective clinical studies that incorporation of Lp-PLA2 testing in cardiovascular risk assessment improves clinical outcomes. At the present time, measurements of Lp-PLA2 are not a component of the guidelines developed by the National Cholesterol Education Program Adult Treatment Panel III. Review History February 2005 March 2007 February 2008 Medical Advisory Council initial approval Added to Scientific Rationale and lipoprotein CPT codes Update. Added additional information in the Scientific Rationale regarding the NMR LipoProfile test. This test refers to Cardiac Risk Assessment-Laboratory Tests Oct 15 37

February 2009 March 2009 July 2009 March 2010 October 2010 May 2011 July 2011 July 2012 September 2012 June 2013 October 2013 October 2014 October 2015 lipoprotein remnants, intermediate density lipoproteins (IDL) and small density lipoproteins, which remain investigational for all Commercial Members. A local Medicare carrier does cover this test so it must be covered for all Medicare Advantage members who reside in the local area in which coverage is applicable. Update. Added measurement Lp-PLA2 as medically necessary for use in high-risk individuals. (Recommendation of American College of Cardiology [ACC] and the American Diabetes Association [ADA]. Medical Advisory Council wanted this to be re-reviewed with evidence-based articles. Update. Evidence-based research reflected that Lp-PLA2 may improve risk prediction for LDL and recurrent stroke, but testing has not been proven to improve health outcomes. These tests are therefore considered investigational and therefore not medically necessary. The previous local Medicare Article for Lipid Profile/Cholesterol Testing Coding Guidelines for LCD L3612, has been removed from policy statement. (A41043) has been retired effective 7/17/08. Added VAP (Vertical Auto Profile) Cholesterol Test as investigational, since the long-term safety and efficacy has not been determined. Update. No revisions. Codes reviewed. Update. Added Corus CAD gene expression test as not medically necessary. Added Medicare Table. Code updates Update no revisions Update - no revisions Update added Palmetto website to the Medicare table for coverage of Corus CAD for Medicare members who meet the criteria. Update no revisions. Codes updated. Update no revisions. Update no revisions. Codes updated. Update no revisions. Codes updated. This policy is based on the following evidence-based guidelines: 1. American Heart Association. Inflammation, Heart Disease and Stroke: The Role of C-Reactive Protein. January 23, 2005. 2. U.S. Department of Health and Human Services, National Institutes of Health (NIH), National Heart, Lung and Blood Institute (NHLBI). Third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Final Report. NIH Publication No. 02-5215. Bethesda, MD: NIH; September 2002. Accessed at: http://circ.ahajournals.org/cgi/reprint/106/25/3143 3. AHA/CDC Scientific Statement. Markers of inflammation of cardiovascular disease. Application to clinical practice: a statement for healthcare professionals from the Centers of Disease Control and Prevention and the American Heart Association. Circulation 2003:107:499-532. Accessed at: http://circ.ahajournals.org/cgi/content/full/107/3/499 Cardiac Risk Assessment-Laboratory Tests Oct 15 38

4. A Statement for Healthcare Professionals From the AHA Task Force on Risk Reduction. Primary Prevention of Coronary Heart Disease: Guidance From Framingham. Circulation. 1998;97:1876-1887. 5. Goldstein LB, Adams R, Alberts MJ, et al. Primary Prevention of Ischemic Stroke: A Guideline from the American Heart Association/ American Stroke Association Stroke Council: co-sponsored by the Atherosclerotic Peripheral Vascular Disease Interdisciplinary Working Group; Cardiovascular Nursing Council; Clinical Cardiology Council; Nutrition, Physical Activity, and Metabolism Council; and the Quality of Care and Outcomes Research Interdisciplinary Working Group: The American Academy of Neurology affirms the value of this guideline. Circulation 2006; 113;e873-e923. Available at: http://circ.ahajournals.org/cgi/content/full/113/24/e873 6. HAYES Search and Summary. Lipoprotein Subclass Quantification Using NMR LipoProfile Test (LipoScience Inc.) for Atherosclerosis/Coronary Artery Disease (CAD). April 2007. Updated April 3, 2009. Archived May 7, 2010. 7. Hayes Technology Brief. Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Test (PLAC Test) (diadexus Inc.) for Prediction of Ischemic Stroke. July 18, 2007. Updated July 31, 2009. Updated July 22, 2010. Archived January 31, 2011. 8. Hayes. Apolipoprotein B Testing for Screening, Diagnosis, and Management of Dyslipidemia and Cardiovascular Disease. November 15, 2000. Archived September 20, 2006. 9. Hayes. Search & Summary. Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Test for the Prediction of Coronary Heart Disease. September 2, 2008. 10. Hayes Health Technology Directory. Lipoprotein-Associated Phospholipase Testing for Coronary Heart Disease Risk Assessment in Healthy or Asymptomatic Adults. Dec 2010. Updated January 17, 2014. 11. Greenland P, Alpert JS, Beller GA, et al. 2010 ACCF/AHA guideline for assessment of cardiovascular risk in asymptomatic adults: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2010 Dec 14;56(25):e50-103. Available at: http://circ.ahajournals.org/cgi/reprint/122/25/2748.pdf 12. Hayes Health Technology Directory. Lipoprotein-Associated Phospholipase Testing for Stroke Assessment in Adults. January 31, 2011, Updated January 9, 2012. Updated January 17, 2014. Updated January 6, 2015. 13. Perk J, De Backer G, Gohlke H, et al.; ESC Committee for Practice Guidelines (CPG. European Guidelines on cardiovascular disease prevention in clinical practice (version 2012): The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by representatives of nine societies and by invited experts). Developed with the special contribution of the European Association for Cardiovascular Prevention & Rehabilitation (EACPR). Eur Heart J. 2012;33(13):1635-1701. 14. Hayes. Genetic Test Evaluation Overview (GTE). Corus CAD. (CardioDx Inc.). June 17, 2011. Updated September 2014. 15. Hayes. Genetic Test Evaluation Overview (GTE). Fibrinogen-Beta (FGB) c.- 455G>A Polymorphism Testing for Cardiovascular Disease (Rosalind Franklin University- Clinical Immunology Laboratory). August 7, 2012. 16. Standards of Medical Care in Diabetes. Diabetes Care, Volume 36, Supplement 1, January 2013. Available at: http://care.diabetesjournals.org/content/36/supplement_1/s11.full.pdf+html 17. Ashley EA, Hershberger RE, Caleshu C, et al. Genetics and cardiovascular disease: a policy statement from the American Heart Association. Circulation. 2012;126(1):142-57. Cardiac Risk Assessment-Laboratory Tests Oct 15 39

18. Dolor MR, Melloni C, Chatterjee R, et al. Noninvasive Tecnologies for the Diagnosis of Coronary Artery Disease in Women. Comparative Effectiveness Review No. 58. AHRQ. 2012;12. 19. Fihn SD, Gardin JM, Abrams J, et al. 2012 ACCF/AHA/ACP/AATS/PCNA/SCAI/STS Guideline for the diagnosis and management of patients with stable ischemic heart disease: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, and the American College of Physicians, American Association for Thoracic Surgery, Preventive Cardiovascular Nurses Association, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons. J Am Coll Cardiol. 2012;60(24):e44-e164. 20. Thomas GS, Szilard V, McPherson JA, et al. A Blood-Based Gene Expression Test for Obstructive Coronary Artery Disease Tested in Symptomatic Non-diabetic Patients Referred for Myocardial Perfusion Imaging. The COMPASS Study. American Heart Association. Circ Cardiovasc Genet 2013;6;154-162. 21. Fleisher LA, Fleischmann KE, Auerbach AD, et al. 2014 ACC/AHA Guideline on Perioperative Cardiovascular Evaluation and Management of Patients Undergoing Noncardiac Surgery: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014 Jul, and Circulation 2014. 22. National Heart, Lung, and Blood Institute (NHLBI). CVD Risk Calculator >> Risk Assessment Tool for Estimating Your 10-year Risk of Having a Heart Attack. Updated May 2013. Available at: http://cvdrisk.nhlbi.nih.gov/calculator.asp. 23. Hayes GTE Overview. Corus CAD. September 2014. 24. Jonas DE, Feltner C, Amick HR, et al. Screening for asymptomatic carotid artery stenosis: A systematic review and meta-analysis for the U.S. Preventive Services Task Force. Ann Intern Med. 2014;161(5):336-346. 25. Kim J, McEvoy JW, Nassir K, et al. Cardiovascular Prospective. Critical Review of High-Sensitivity C-Reactive Protein and Coronary Artery Calcium for the Guidance of Statin Allocation. Head-to-Head Comparison of the JUPITER and St. Francis Heart Trials. September 16, 2015. Available at: http://circoutcomes.ahajournals.org/content/7/2/315.full.pdf+html 26. Lackland DT, Roccella EJ, Deutsch AF, et al, American Heart Association Stroke Council. Factors influencing the decline in stroke mortality: a statement from the American Heart Association/American Stroke Association. Stroke. 2014; 45:315-53. 27. National Institute for Health and Care Excellence (NICE). Lipid modification: Cardiovascular risk assessment and the modification of blood lipids for the primary and secondary prevention of cardiovascular disease. NICE Clinical Guideline 181. July 2014 (modified January 2015). (Guideline updates and replaces NICE guideline CG67 and NICE technology appraisal guidance 94). 28. Stone NJ, Robinson J, Lichtenstein AH, et al. 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults. J Am Coll Cardiol. 2014; 63(25). References Update October 2015 1. Kara H, Akinci M, Degirmenci S, et al. High-sensitivity C-reactive protein, lipoprotein-related phospholipase A2, and acute ischemic stroke. Neuropsychiatr Dis Treat. 2014 Aug 6;10:1451-7. doi: 10.2147/NDT.S67665. ecollection 2014. 2. Katan M, Moon YP, Paik MC, et al. Lipoprotein-associated phospholipase A2 is associated with atherosclerotic stroke risk: the Northern Manhattan Study. PLoS One. 2014 Jan 9;9(1):e83393. doi: 10.1371/journal.pone.0083393. ecollection 2014. Cardiac Risk Assessment-Laboratory Tests Oct 15 40

3. Kleber ME, Siekmeier R, Delgado G, et al. C-reactive protein and lipoproteinassociated phospholipase A2 in smokers and nonsmokers of the Ludwigshafen Risk and Cardiovascular Health study. Advances in experimental medicine and biology. 2015;832:15-23. 4. LeFevre ML. Screening for Asymptomatic Carotid Artery Stenosis: U.S. Preventive Services Task Force Recommendation Statement Screening for Asymptomatic Carotid Artery Stenosis. Clinical Guidelines 2 September 2014. Ann Intern Med. 2014;161(5):356-362. doi:10.7326/m14-1333. Available at: http://annals.org/article.aspx?articleid=1886690 5. Wasserheil -Smoller S, McGinn A, Allison M, et al. Improvement in stroke risk prediction: role of C-reactive protein and lipoprotein-associated phospholipase A2 in the women's health initiative. Int J Stroke. 2014 Oct;9(7):902-9. doi: 10.1111/j.1747-4949.2012.00860.x. Epub 2012 Oct 23. 6. White HD, Held, C, Stewart, R, et al. Darapladib for preventing ischemic events in stable coronary heart disease. The New England journal of medicine. 2014 May 1;370(18):1702-11. References Update October 2014 1. Guanglin Cui, Zongzhe Li, Rui Li, et al. A Functional Variant in APOA5/A4/C3/A1 Gene Cluster Contributes to Elevated Triglycerides and Severity of CAD by Interfering With MicroRNA 3201 Binding Efficiency. J Am Coll Cardiol. 2014;64(3):267-277. doi:10.1016/j.jacc.2014.03.050. 2. Herman L, Froelich J, Kanelos D, et al. Utility of a genomic-based, personalized medicine test in patients presenting with symptoms suggesting coronary artery disease. J Am Board Fam Med. 2014;27(2):258-267. 3. Hochheiser LI, Juusola JL, Monane M, Ladapo JA. Economic utility of a bloodbased genomic test for the assessment of patients with symptoms suggestive of obstructive coronary artery disease. Popul Health Manag. 2014 Feb 25. [Epub ahead of print]. 4. Ladapo JA, Lyons H, Yau M, et al. Enhanced assessment of chest pain and related symptoms in the primary care setting through the use of a novel personalized medicine genomic test: Results from a prospective registry study. Am J Med Qual. 2014 May 5. [Epub ahead of print]. 5. O'Donoghue ML, Morrow DA, Tsimikas S, et al. Lipoprotein(a) for risk assessment in patients with established coronary artery disease. J Am Coll Cardiol. 2014;63(6):520-527. 6. Patel RS, Asselbergs FW, Quyyumi AA, et al. Genetic variants at chromosome 9p21 and risk of first versus subsequent coronary heart disease events: a systematic review and meta-analysis. J Am Coll Cardiol. 2014 Jun 3;63(21):2234-45. doi: 10.1016/j.jacc.2014.01.065. Epub 2014 Mar 7. 7. Roncarati R, Viviani Anselmi C, Losi MA, et al. Circulating mir-29a, among other up-regulated micrornas, is the only biomarker for both hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy. J Am Coll Cardiol. 2014;63 (9):920-927. 8. Voros S, Elashoff MR, Wingrove JA, et al. A peripheral blood gene expression score is associated with atherosclerotic plaque burden and stenosis by cardiovascular CT-angiography: Results from the PREDICT and COMPASS studies. Atherosclerosis. 2014;233(1):284-290. References Update October 2013 1. Clinicaltrials.gov. A Registry to Evaluate Patterns of Care Associated With the Use of Corus CAD in Real World Clinical Care Settings (PRESET). Cardiac Risk Assessment-Laboratory Tests Oct 15 41

ClinicalTrials.gov Identifier:NCT01677156. August 29, 2012. Available at: http://www.clinicaltrials.gov/ct2/show/nct01677156 2. Clinicaltrials.gov. Investigation of a Novel Gene Expression Test for the Diagnosis of Obstructive Coronary Artery Disease on Physician's Practice Pattern. ClincialTrials.gov Identifier: NCT01557855. February 2013. Available at: http://www.clinicaltrials.gov/ct2/show/nct01557855 3. Clinicaltrials.gov. Primary Care Providers Use of a Gene Expression Test in Coronary Artery Disease Diagnosis (IMPACT-PCP). ClinicalTrials.gov Identifier: NCT01594411. February 7, 2013. Available at: http://www.clinicaltrials.gov/ct2/show/nct01594411?term=primary+care+provi ders+use+of+a+gene+expression+test+in+coronary+artery+disease+diagno sis&rank=1 4. Conlin L, Mouton M, Wilson L, et al. The Use of a Personalized Gene Expression Test to Improve Decision Making in the Evaluation of Patients with Suspected Coronary Artery Disease. J Gen Intern Med. 2012;27:S540-1. 5. Doring Y, Noels H, Weber C. The use of high-throughput technologies to investigate vascular inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol. 2012;32(2):182-95. 6. Elashoff MR, Nuttall R, Beineke P, et al. Identification of factors contributing to variability in a blood-based gene expression test. PLoS One. 2012;7(7):e40068. 7. McPherson JA, Davis K, Yau M, et al. The Clinical Utility of Gene Expression Testing on the Diagnostic Evaluation of Patients Presenting to the Cardiologist With Symptoms of Suspected Obstructive Coronary Artery Disease: Results From the IMPACT (Investigation of a Molecular Personalized Coronary Gene Expression Test on Cardiology Practice Pattern) Trial. Crit Pathw Cardiol. 2013;12(2):37-42. 8. MolDx Corus CAD Test Coding and Billing Guidelines. 2013. Available at: http://www.palmettogba.com/palmetto/providers.nsf/docscat/providers~jurisdi ction%201%20part%20b~browse%20by%20topic~moldx~covered%20tests~ 8WXQ5R5416?open&navmenu=%7C%7C 9. Rozanski A, Gransar H, Hayes SW, et al. Temporal trends in the frequency of inducible myocardial ischemia during cardiac stress testing: 1991 to 2009. J Am Coll Cardiol. 2013;61(10):1054-65. 10. Vargas J, Lima JAC, Kraus WE, et al. Use of the Corus CAD Gene Expression Test for Assessment of Obstructive Coronary Artery Disease Likelihood in Symptomatic Non-Diabetic Patients. PLOS. Currents Evidence on Genomic Tests. 2013 Aug 26. Edition 1. doi:10.1371. 11. Veenstra DL, Piper M, Haddow JE, et al. Improving the efficiency and relevance of evidence-based recommendations in the era of whole-genome sequencing: an EGAPP methods update. Genet Med. 2012. Epub ahead of print. September 6, 2012. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22955111. References Update June 2013 1. Benetos A, Gautier S, Labat C, et al. Mortality and Cardiovascular Events Are Best Predicted by Low Central/Peripheral Pulse Pressure Amplification But Not by High Blood Pressure Levels in Elderly Nursing Home Subjects: The PARTAGE (Predictive Values of Blood Pressure and Arterial Stiffness in Institutionalized Very Aged Population) Study. Journal of the American College of Cardiology. October 2012. 2. Rosenson RS. Lipoprotein classification; metabolism; and role in atherosclerosis. UpToDate. October 22, 2012. Cardiac Risk Assessment-Laboratory Tests Oct 15 42

References Update September 2012 1. Ahmed MS, Ji JZ, Meng QH. Lipoprotein-associated phospholipase A2: how effective as a risk marker of cardiovascular disease and as a therapeutic target? Inflamm Allergy Drug Targets. 2011 Aug 1;10(4):236-46. 2. CardioDx. CardioDx Announces Medicare Coverage for Corus CAD Gene Expression Test for the Diagnosis of Obstructive Coronary Artery Disease. Press Release. 2012. Available at: http://www.cardiodx.com/aboutcardiodx/newsroom/press-releases/medicare-coverage-corus-cad 3. Clinicaltrials.gov. Personalized Risk Evaluation and Diagnosis In the Coronary Tree (PREDICT). CardioDX. 2012. ClinicalTrials.gov Identifier: NCT00500617. Available at: http://clinicaltrials.gov/ct2/show/nct00500617 4. Clinicaltrials.gov. Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS). ClinicalTrials.gov Identifier: NCT01117506. March 16, 2012. Available at: http://clinicaltrials.gov/ct2/show/nct01117506?term=compass&rank=2 5. Clinicaltrials.gov. Investigation of a Molecular Personalized Coronary Gene Expression Test on Cardiology Practice Pattern (IMPACT-CARD). ClinicalTrials.gov Identifier: NCT01251302. March 16, 2012. Available at: http://clinicaltrials.gov/ct2/show/nct01251302?term=impact+- +Corus+CAD&rank=1 6. Colley KJ, Wolfert RL, Cobble ME. Lipoprotein associated phospholipase A(2): role in atherosclerosis and utility as a biomarker for cardiovascular risk. EPMA J. 2011 Mar;2(1):27-38. Epub 2011 Mar 10. 7. Constantinides A, van Pelt LJ, van Leeuwen JJ, et al. Carotid intima media thickness is associated with plasma lipoprotein-associated phospholipase A2 mass in nondiabetic subjects but not in patients with type 2 diabetes. Eur J Clin Invest. 2011 Aug;41(8):820-7. Epub 2011 Jan 31. 8. Douglas PD, Poppas A. Determinants and management of cardiovascular risk in women. UpToDate. June 6, 2012. 9. Elashoff MR, Wingrove JA, Beineke P, et al. Development of a blood-based gene expression algorithm for assessment of obstructive coronary artery disease in non-diabetic patients. BMC Med Genomics. 2011;4:26. 10. Florentin M, Liberopoulos EN, Moutzouri E, et al. The effect of simvastatin alone versus simvastatin plus ezetimibe on the concentration of small dense lowdensity lipoprotein cholesterol in subjects with primary hypercholesterolemia. Curr Med Res Opin. 2011 Mar;27(3):685-92. Epub 2011 Jan 27. 11. Ginsberg GS. Goldman: Goldman's Cecil Medicine, 24th ed. 2011 Saunders, An Imprint of Elsevier. Application of Molecular Technologies to Clinical Medicine. 12. Gong HP, Du YM, Zhong LN, et al. Plasma lipoprotein-associated phospholipase A2 in patients with metabolic syndrome and carotid atherosclerosis. Lipids Health Dis. 2011 Jan 19;10:13. 13. Kavousi M, Elias-Smale S, Rutten JH, et al. Evaluation of newer risk markers for coronary heart disease risk classification: A cohort study. Ann Intern Med. 2012;156(6):438-444 14. Lansky A, Elashoff MR, Ng V, et al. A gender-specific blood-based gene expression score for assessing obstructive coronary artery disease in nondiabetic patients: Results of the Personalized Risk Evaluation and Diagnosis in the Coronary Tree (PREDICT) Trial. American Heart Journal. (24 July 2012). 15. Martin SS, Qasim AN, Wolfe M, et al. Comparison of high-density lipoprotein cholesterol to apolipoprotein A-I and A-II to predict coronary calcium and the effect of insulin resistance. Am J Cardiol. 2011 Feb 1;107(3):393-8. Cardiac Risk Assessment-Laboratory Tests Oct 15 43

16. Otvos JD, Mora S, Shalaurova I, et al. Clinical implications of discordance between low-density lipoprotein cholesterol and particle number. J Clin Lipidol. 2011 Mar-Apr;5(2):105-13. 17. Palmetta GBA, Jurisdiction 11 Part B. Corus CAD Test Coding and Billing Guidelines. 8/7/2012. 18. Rosenberg S, Elashoff MR, Beineke P, et al. Multicenter validation of the diagnostic accuracy of a blood-based gene expression test for assessing obstructive coronary artery disease in nondiabetic patients. Ann. Intern. Med. 2010;153(7):425-434. References Update July 2012 1. Berry JD, Dyer A, Cai X, et al. Lifetime risks of cardiovascular disease. N Engl J Med 2012; 366:321. 2. Centers for Disease Control and Prevention (CDC). Vital signs: prevalence, treatment, and control of high levels of low-density lipoprotein cholesterol-- United States, 1999-2002 and 2005-200. MMWR Morb Mortal Wkly Rep 2011; 60:109. 3. Chaitman BR. Bonow: Braunwald's Heart Disease. A Textbook of Cardiovascular Medicine, 9th ed. 2011 Saunders, An Imprint of Elsevier. 4. Ferraro S, Santagostino M, Marano G, et al. The prognostic value of plasma fibrinogen concentrations of patients with ST-elevation myocardial infarction and treated by primary percutaneous coronary intervention: A cautionary message. Scand J Clin Lab Invest. 2012 Apr 10. 5. Garg A. What is the role of alternative biomarkers for coronary heart disease? Clin Endocrinol (Oxf). 2011 Sep;75(3):289-93. doi: 10.1111/j.1365-2265.2011.04045.x. 6. Hirschler V, Meroño T, Maccallini G, et al. Association of lipoprotein-associated phospholipase A2 activity with components of the metabolic syndrome in apparently healthy boys. Cardiovasc Hematol Agents Med Chem. 2011 Apr 1;9(2):78-83. 7. Neogi T, Terkeltaub R, Ellison RC, et al. Serum urate is not associated with coronary artery calcification: the NHLBI Family Heart Study. Rheumatol. 2011 Jan;38(1):111-7. Epub 2010 Oct 1. 8. Soh J, Josekutty J, Mahmood Hussain M. LIPOPROTEINS, APOLIPOPROTEINS, AND RELATED PROTEINS. McPherson: Henry's Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. 2011 Saunders, An Imprint of Elsevier 9. Morrow DA, C-reactive protein in cardiovascular disease. UpToDate. July 14, 2011. 10. Rosenson RS. Screening guidelines for dyslipidemia. UpToDate. April 25, 2011. 11. Rosenson RS, Brewer HB Jr, Chapman MJ, et al. HDL measures, particle heterogeneity, proposed nomenclature, and relation to atherosclerotic cardiovascular events. Clin Chem 2011; 57:392. 12. Rosenson RS. Stein JH, Durrington P. Lipoproteins and Cardiovascular Disease. UpToDate. July 20, 2011. Updated April 29, 2013. 13. Smith TW. Morgan JP. Actions of angiotensin II on the heart. UpToDate. July 8, 2011. 14. Stamler J, Stamler R, Neaton JD, et al. Low risk-factor profile and long-term cardiovascular and noncardiovascular mortality and life expectancy: findings for 5 large cohorts of young adult and middle-aged men and women. JAMA 1999; 282:2012. 15. Wilson PWF. Estimation of cardiovascular risk in an individual patient without known cardiovascular disease. UpToDate. May 2, 2012. Cardiac Risk Assessment-Laboratory Tests Oct 15 44

16. Yanowitz SG. Screening for coronary heart disease. UpToDate. May 16, 2012. Updated October 12. 2012. 17. Zheng GH, Chen HY, Xiong SQ, et al. Lipoprotein-associated phospholipase A2 gene V279F polymorphisms and coronary heart disease: a meta-analysis. Mol Biol Rep. 2011 Aug;38(6):4089-99. Epub 2010 Nov 24. References Update July 2011 1. Chiang CW, Santos RD, Waters DD, et al. Reaching C-Reactive Protein and Low- Density Lipoprotein Cholesterol Goals in Dyslipidemic Patients (from the Lipid Treatment Assessment Project [L-TAP] 2). 2. Hatoum IJ, Cook NR, Nelson JJ, et al. Lipoprotein-associated phospholipase A2 activity improves risk discrimination of incident coronary heart disease among women. Am Heart J. 2011 Mar;161(3):516-22 3. Jenny NS, Solomon C, Cushman M, et al. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and risk of cardiovascular disease in older adults: results from the Cardiovascular Health Study. Atherosclerosis. 2010 Apr;209(2):528-32 4. Li N, Li S, Yu C, Gu S. Plasma Lp-PLA2 in acute coronary syndrome: association with major adverse cardiac events in a community-based cohort. Postgrad Med. 2010 Jul;122(4):200-5. 5. Lim LS, Haq N, Mahmood S, et al. Atherosclerotic cardiovascular disease screening in adults: American College Of Preventive Medicine position statement on preventive practice. Am J Prev Med. 2011 Mar;40(3):381.e1-10. 6. Lp-PLA(2) Studies Collaboration, Thompson A, Gao P, et al. Lipoproteinassociated phospholipase A(2) and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies. Lancet. 2010 May 1;375(9725):1536-44. 7. Suzuki T, Solomon C, Jenny NS, et al. Lipoprotein-associated phospholipase A(2) and risk of congestive heart failure in older adults: the Cardiovascular Health Study. Circ Heart Fail. 2009 Sep;2(5):429-36. 8. Wannamethee SG, Welsh P, Lowe GD, et al. N-terminal pro-brain natriuretic Peptide is a more useful predictor of cardiovascular disease risk than C-reactive protein in older men with and without pre-existing cardiovascular disease. J Am Coll Cardiol. 2011 Jun 28;58(1):56-64. References Update October 2010 1. Lansky AJ, Elashoff MR, Sehnert AJ, et al. Myocardial Perfusion Imaging Performance in the PREDICT multicenter trial; Gender Specific Abalyses and Comparison with the CORUS CAD Gene Expression Score. J. Am. Coll. Cardiol. 2010;55;A186.E1739. 2. Clinicaltrials.gov. Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS). May 10, 2010. ClinicalTrials.gov Identifier: NCT01117506. Available at: http://clinicaltrials.gov/ct2/show?term=cardiodx&rank=1 3. Lloyd-Jones D, Adams R, Carnethon M, et al. for the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics. 2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2009;119(3):480-486. 4. Doesch C, Seeger A, Hoevelborn T, et al. Adenosine stress cardiac magnetic resonance imaging for the assessment of ischemic heart disease. Clin Res Cardiol. 2008;97 :905-912. Cardiac Risk Assessment-Laboratory Tests Oct 15 45

5. Nandalur KR, Dwamena BA, Choudhri AF, et al. Diagnostic performance of stress cardiac magnetic resonance imaging in the detection of coronary artery disease: a meta-analysis. J Am Coll Cardiol. 2007;50(14):1343-1353. References Update March 2010 1. Angadi SS. Pre-Exercise Cardiology Screening Guidelines for Asymptomatic Patients with Diabetes. Clinics in Sports Medicine - Volume 28, Issue 3 (July 2009). 2. Schuerch C, Selna M, Jones J. Laboratory Clinical Effectiveness: Pathologists Improving Clinical Outcomes. Laboratory Clinical Effectiveness: Pathologists Improving Clinical Outcomes. Clinics in Laboratory Medicine - Volume 28, Issue 2 (June 2008). 3. Persson M, Berglund G, Nelson JJ, et al. Lp-PLA2 activity and mass are associated with increased incidence of ischemic stroke: a population-based cohort study from Malmö, Sweden. Atherosclerosis. 2008;200(1):191-198. 4. Wassertheil-Smoller S, Kooperberg C, McGinn AP, et al. Lipoprotein-associated phospholipase A2, hormone use, and the risk of ischemic stroke in postmenopausal women. Hypertension. 2008;51(4):1115-1122. 5. Sabatine MS, Morrow DA, O'Donoghue M, et al.; PEACE Investigators. Prognostic utility of lipoprotein-associated phospholipase A2 for cardiovascular outcomes in patients with stable coronary artery disease. Arterioscler Thromb Vasc Biol. 2007;27(11):2463-2469. Available at: http://atvb.ahajournals.org/cgi/content/full/27/11/2463 6. Elkind MS, Tai W, Coates K, et al. High-sensitivity C-reactive protein, lipoproteinassociated phospholipase A2, and outcome after ischemic stroke. Arch Intern Med. 2006;166(19):2073-2080. Available at: http://archinte.amaassn.org/cgi/content/full/166/19/2073 7. Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-associated phospholipase A2, high-sensitivity C-reactive protein, and risk for incident ischemic stroke in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Arch Intern Med. 2005;165(21):2479-2484. Available at: http://archinte.ama-assn.org/cgi/content/full/165/21/2479 References Update July 2009 1. Lau JF, Smith DA. Endocrinol Metab Clin North Am - 01-MAR-2009; 38 (1): 1-31. 2. Kulkarni KR. Cholesterol profile measurement by vertical auto profile method. 1: Clin Lab Med. 2006 Dec; 26 (4): 787-802. 3. Ensign W, Hill N, Heward CB. Disparate LDL phenotypic classification among 4 different methods assessing LDL particle characteristics. 1: Clin Chem. 2006 Sep; 52(9): 1722-7. Epub 2006 Jun 1. References Update March 2009 1. Nambi V, Hoogeveen RC, Chanbless L, et al. Lipoprotein-Associated Phospholipase A2 and High-Sensitivity C-Reactive Protein Improve the Stratification of Ischemic Stroke Risk in the Atherosclerosis Risk in Communities (ARIC) Study. Arch Intern Med. January 26, 2009. 2. Daniels L, Laughlin G, Sarno M, et al. Lipoprotein-associated phospholipase A2 is an independent predicator of incident coronary heart disease in an apparently healthy older population: The Rancho Bernardo Study. (2008). Journal of American College of Cardiology, 4, 51 (9), 913-919. Cardiac Risk Assessment-Laboratory Tests Oct 15 46

3. Persson M, Berglund G, Nelson JJ, et al. Lp-PLA2 Activity and Mass are Associated with Increased Incidence of Ischemic Stroke. A Population-Based Cohort Study from Malmo, Sweden. Atherosclerosis 2008. 4. Wassertheil-Smoller S, Kooperberg C, McGinn AP, et al. Lipoprotein-Associated Phospholipase A2, Hormone Use, and the Risk of Ischemic stroke in Postmenopausal Women. February 8,2008. 5. Ridker PM, Danielson E, Francisco MIA, et al. Rosuvastatin to Prevent Vascular Events in Men and Women with Elevated C-Reactive Protein. The Jupiter Study Group. New England Journal of Medicine. Volume 359:2195-2207, November 20, 2008, Number 21. 6. Elkind MSV, Tai W, Coates K, et al. High Sensitivity C-Reactive Protein, Lipoprotein-Assocated Phospholipase A2, and Outcome After Ischemic Stroke. Arch Intern Med. 2006; 166:2073-2080. 7. Kastelein JP, Wedel MK, Baker BF, et al. Potent Reduction of Apolipoprotein B and Low-Density Lipoprotein Cholesterol by Short-Term Administration of an Antisense Inhibitor of Apolipoprotein B. Circulation. 2006; 114:1729-1735.) 8. Marcovina S, Packard CJ. Measurement and meaning of apolipoprotein AI and apolipoprotein B plasma levels. Intern Med. 2006 May; 259(5): 437-46. 9. Barter PJ, Ballantyne CM, Carmena R, et al. Apo B versus cholesterol in estimating cardiovascular risk and in guiding therapy: report of the thirtyperson/ten-country panel. J Intern Med. 2006 Mar;259(3):247-58. 10. Thompson A, Danesh J. Associations between apolipoprotein B, apolipoprotein AI, the apolipoprotein B/AI ratio and coronary heart disease: a literature-based meta-analysis of prospective studies. J Intern Med. 2006 May;259(5):481-92. 11. Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-Associated Phospholipase A2, High-Sensitivity C-reactive protein, and Risk for Incident Ischemic Stroke in Middle-aged Men and Women in the Atherosclerosis Risk in Communities (ARIC) Study. Arch Intern Med. Volume 165, November 28, 2005. 12. Hok-Hay SO, van der Meer IM, Hofman A, et al. Lipoprotein-Associated Phospholipase A2 Activity is Associated With Risk of Coronary Heart Disease and Ischemic Stroke. Circulation, 2005; 111:570-575. 13. McNeill AM, et al. The metabolic syndrome and 11-year risk of incident cardiovascular disease in the atherosclerosis risk in communities study. ARIC Study. Diabetes Care 2005;28:385-90. References Update February 2009 1. Davidson MH, Corson MA, Alberts MJ, et al. Consensus Panel Recommendation for Incorporating Lipoprotein-Associated Phospholipase A2 Testing into Cardiovascular Disease Risk Assessment Guidelines. The American Journal of Cardiology - Volume 101, Issue 12A (June 2008) 2. Brunzell JD, Davidson M, Furberg CD, et al. Lipoprotein management in patients with cardiometabolic risk. Consensus statement from the American Diabetes Association and the American College of Cardiology Foundation. Diabetes Care. 2008;31 (4):811-822. 3. Weintraub HS. Identifying the vulnerable patient with rupture-prone plaque. Am J Cardiol. 2008 Jun 16;101(12A):3F-10F. 4. Schmidt EB, Koenig W, Khuseyinova N, et al. Lipoprotein-associated phospholipase A2 concentrations in plasma are associated with the extent of coronary artery disease and correlate to adipose tissue levels of marine n-3 fatty acids. Atherosclerosis. 196(1):420-4, 2008 Jan. 5. Robins SJ, Collins D, Nelson JJ, et al. Cardiovascular events with increased lipoprotein-associated phospholipase A(2) and low high-density lipoprotein- Cardiac Risk Assessment-Laboratory Tests Oct 15 47

cholesterol: the Veterans Affairs HDL Intervention Trial. Arterioscler Thromb Vasc Biol. 2008 Jun;28(6):1172-8. Epub 2008 Mar 20. 6. Mockel M, Danne O, Muller R, et al. Development of an optimized multimarker strategy for early risk assessment of patients with acute coronary syndromes. Clinica Chimica Acta. 393(2)(pp 103-109), 2008. Date of Publication: 17 Jul 2008. 7. Khuseyinova N, Greven S, Ruckerl R, et al. Variability of serial lipoproteinassociated phospholipase A2 measurements in post myocardial infarction patients: results from the AIRGENE Study Center Augsburg. Clinical Chemistry. 54(1):124-30, 2008 Jan. 8. Furberg CD, Nelson JJ, Solomon C, et al. Distribution and correlates of lipoprotein-associated phospholipase A2 in an elderly cohort: the Cardiovascular Health Study. J Am Geriatr Soc. 2008 May;56(5):792-9. Epub 2008 Mar 21. 9. Daniels LB, Laughlin GA, Sarno MJ, et al. Lipoprotein-associated phospholipase A2 is an independent predictor of incident coronary heart disease in an apparently healthy older population: the Rancho Bernardo Study. Journal of the American College of Cardiology. 51(9):913-9, 2008 Mar 4. 10. Corson MA, Jones PH, Davidson MH. Review of the Evidence for the Clinical Utility of Lipoprotein-Associated Phospholipase A2 as a Cardiovascular Risk Marker. American Journal of Cardiology. 101(12 SUPPL.)(pp S41-S50), 2008. Date of Publication: 16 Jun 2008. 11. Anderson JL. Lipoprotein-Associated Phospholipase A2: An Independent Predictor of Coronary Artery Disease Events in Primary and Secondary Prevention. American Journal of Cardiology. 101(12 SUPPL.)(pp S23-S33), 2008. Date of Publication: 16 Jun 2008. References Update February 2008 1. Kathiresan S, Otvos JD, Sullivan LM, et al. Increased small low-density lipoprotein particle number: a prominent feature of the metabolic syndrome in the Framingham Heart Study. Circulation. 2006;113(1):20-29. 2. Ensign W, Hill N, Heward CB. Disparate LDL phenotypic classification among 4 different methods assessing LDL particle characteristics. Clin Chem. 2006;52(9):1722-1727. 3. Ballantyne CM, Hoogeveen RC. Role of lipid and lipoprotein profiles in risk assessment therapy. Am Heart J. 2003 Aug;146(2):227-33. 4. Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) final report. Circulation. 2002;106(25):3143-3421. 5. Centers for Medicare and Medicaid Services (CMS). Local Coverage Determination. Article for Lipid Profile/Cholesterol Testing Coding Guidelines for LCD L3612. (A41043) (Retired) References Update March 2007 1. Folsom AR, Chambless LE, Ballantyne, CM, et al. An assessment of incremental coronary risk prediction using c-reactive protein and other novel risk markers: The Atherosclerosis Risk in Communities Study. Arch Intern Med. 2006; 166:1368-1373. 2. Mitchell et al. High-Sensitivity C-Reactive Protein, Lipoprotein-Associated Phospholipase A2, and Outcome After Ischemic Stroke. Arch Intern Med. 2006;166:2073-2080. Cardiac Risk Assessment-Laboratory Tests Oct 15 48

3. Lloyd-Jones DM, Tian L. Predicting cardiovascular risk: So what do we do now? Arch Intern Med. 2006; 166:1342-1344. 4. O'Donoghue M, Morrow DA, Sabatine MS, ET AL. Lipoprotein-associated phospholipase A2 and its association with cardiovascular outcomes in patients with acute coronary syndromes in the PROVE IT-TIMI 22 (PRavastatin Or atorvastatin Evaluation and Infection Therapy- Thrombolysis In Myocardial Infarction) trial. Circulation. 2006; 113(14):1745-1752. 5. van Vark LC, Kardys I, Bleumink GS, et al. Lipoprotein-associated phospholipase A2 activity and risk of heart failure: The Rotterdam study. Eur Heart J. 2006 Oct;27(19):2346-52. 6. Kardys I, Oei HH, Hofman A, et al. Lipoprotein-associated phospholipase A2 and coronary calcification The Rotterdam Coronary Calcification Study. Atherosclerosis. 2006 May 3. 7. Brilakis ES, McConnell JP, Lennon RJ, Elesber AA, Meyer JG, Perger PB. Association of lipoprotein-associated phospholipase A2 levels with coronary artery disease risk factors, angiographic coronary artery disease, and major adverse events at follow-up. European Heart Journal. 2005;26:137-144. 8. Ballantyne CM, Hoogeveen RC, Bang H, et al. Lipoprotein-Associated Phospholipase A2, High-Sensitivity C-Reactive Protein, and Risk for Incident Coronary Heart Disease in Middle-Aged Men and Women in the Atherosclerosis Risk in Communities (ARIC) Study. Circulation. 2004;109:837-842. 9. Koenig W, Khuseyinova N, Löwel H, Trischler G, Meisinger C. Lipoprotein- Associated Phospholipase A2 Adds to Risk Prediction of Incident Coronary Events by C-Reactive Protein in Apparently Healthy Middle-Aged Men From the General Population: Results From the 14-Year Follow-Up of a Large Cohort From Southern Germany. Circulation. 2004;110:1903-1908. References - Initial 1. Ballantyne CM. Achieving greater reductions in cardiovascular risk: lessons from statin therapy on risk measures and risk reduction. Am Heart J. 2004;148:3S-8. 2. Grundy S, Cleeman JI, Merz CN, et al. Implications of recent clinical trials for the national cholesterol education program adult treatment panel III guidelines. J Am Coll Cardiol. 2004;44:720-732. 3. LaRossa JC, Gotto AM. Past, present and future standards for management of dyslipidemia. Am J Med. 2004;116(6A):3S-8S. 4. Jadhav UM, Kadam NN. Apolipoproteins: correlation with carotid intimamedia thickness and coronary artery disease. J Assoc Physicians India. 2004 May;52:370-5. 5. Ogiwara F, Takahashi M, Ikeda U. Inflammatory markers and cytokines in cardiovascular disease. Rinsho Byori. 2004 Aug;52(8):686-92. Review. Japanese. 6. Bassuk SS, Rifai N, Ridker PM. High-sensitivity C-reactive protein: clinical importance. Curr Probl Cardiol. 2004 Aug;29(8):439-93. 7. Scott J. Pathophysiology and biochemistry of cardiovascular disease. Curr Opin Genet Dev. 2004 Jun;14(3):271-9. 8. Pearson TA, Mensah GA, Alexander RW, et al. Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association. Circulation. 2003;107(3):499-511. 9. Ridiker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and lowdensity lipoprotein cholesterol levels in the prediction of first cardiovascular event. N Engl J Med. 2002;347:1557-65. Cardiac Risk Assessment-Laboratory Tests Oct 15 49

10. Kushner I, Sehgal AR. Is high-sensitivity C-reactive protein an effective screening test for cardiovascular risk? Arch Intern Med 2002;162:867-9. 11. Goldstein JL, Brown MS: Molecular medicine. The cholesterol quartet. Science. 2001 May 18;292(5520):1310-2. 12. Rifai N, Ridker PM. High sensitivity C-reactive protein: a novel and promising marker of coronary heart disease. Clin Chem. 2001;47:403-411. 13. Ridker PM, Stampfer MJ, Rifai N. Novel risk factors for systemic atherosclerosis: a comparison of C-reactive protein, fibrinogen, homocysteine, lipoprotein(a), and standard cholesterol screening as predictors of peripheral arterial disease. JAMA. 2001;285:2481-2485. 14. Ridker PM. High-sensitivity C-reactive protein: potential adjunct for global risk assessment in the primary prevention of cardiovascular disease. Circulation. 2001;103:1813-1818. 15. Rifai N, Ridker PM. Proposed cardiovascular risk assessment algorithm using highsensitivity C-reactive protein and lipid screening. Clin Chem. 2001;47:28-30. 16. Rost NS, Wolf PA, Kase CS, et al. Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: the Framingham Study. Stroke. 2001;32:2575-2579. 17. Germino FW. Using C-reactive protein in practice. Patient Care 2000; April:50-5. 18. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med. 2000;342:836-843. 19. Roivainen M, Viik-Kajander M, Palosuo T, et al. Infections, inflammation, and the risk of coronary heart disease. Circulation 2000;101:252-257. 20. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-Reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836-843. 21. Du Clos TW. Function of C-reactive protein. Ann Med. 2000;32:274-278. 22. Koenig W, Sund M, Frohlich M, et al. C-Reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men. Results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation 1999;99:237 42. 23. Rifai N, Tracy RP, Ridker PM. Clinical efficacy of an automated high sensitivity C- reactive protein assay. Clin Chem 1999;45:2136-2141. 24. Gabay C, Kushner I. Acute-phase proteins and other systematic responses to inflammation. N Engl J Med. 1999;340:448-454. 25. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999;340:115-126. 26. Ridker PM, Buring JE, Shih J, Matias M, Hennekens CH. Prospective study of C- reactive protein and the risk of future cardiovascular events among apparently healthy women. Circulation 1998;98:731-733. 27. Ridker PM, Glynn RJ, Hennekens CH. C-reactive protein adds to the predictive value of total and HDL cholesterol in determining risk of first myocardial infarction. Circulation. 1998;97:2007-2011. 28. Kuller LH, Tracy RP, Shaten J, et al. Relation of C-reactive protein and coronary heart disease in the MRFIT nested case-control study. Am J Epidemiol. 1996;144:537-547. 29. Mendall MA, Patel P, Ballam L, et al. C-reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. BMJ. 1996;312:1061-1065. 30. Curb JD, Abbott RD, MacLean CJ, et al. Age-related changes in stroke risk in men with hypertension and normal blood pressure. Stroke. 1996;27:819-824. Cardiac Risk Assessment-Laboratory Tests Oct 15 50

31. Saikku P, Leinonen M, Tenkanen L, et al. Chronic Chlamydia pneumoniae infection as a risk factor for coronary heart disease in the Helsinki Heart Study. Ann Intern Med 1992;116:273-8. 32. Goldstein JL, Brown MS: Regulation of low-density lipoprotein receptors: implications for pathogenesis and therapy of hypercholesterolemia and atherosclerosis. Circulation. 1987 Sep;76(3):504-7. Important Notice General Purpose. Health Net's National Medical Policies (the "Policies") are developed to assist Health Net in administering plan benefits and determining whether a particular procedure, drug, service or supply is medically necessary. The Policies are based upon a review of the available clinical information including clinical outcome studies in the peer-reviewed published medical literature, regulatory status of the drug or device, evidence-based guidelines of governmental bodies, and evidence-based guidelines and positions of select national health professional organizations. 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