More Evidence that Low-density Lipoprotein Cholesterol and Triglyceride-lowering Genetic Variants Reduce Risk of Coronary Heart Disease

More Evidence that Low-density Lipoprotein Cholesterol and Triglyceride-lowering Genetic Variants Reduce Risk of Coronary Heart Disease

By Kevin C Maki, PhD and Mary R Dicklin, PhD

 

The total cholesterol concentration in circulation is comprised of cholesterol carried by three main types of lipoproteins:  low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL).  VLDL particles are the main carriers of triglycerides (TG), and the Friedewald equation estimates the VLDL cholesterol (VLDL-C) level in mg/dL as TG/5.  LDL and VLDL particles each contain a single molecule of apolipoprotein B (Apo B).  Non-HDL cholesterol (non-HDL-C) is the sum of the cholesterol carried by all particles that contain Apo B, i.e., LDL-C + VLDL-C.  Note for purists:  this ignores a quantitatively small contribution of cholesterol carried by chylomicron remnants and it includes cholesterol carried by lipoprotein (a) particles, which are generally in the LDL density range.

 

Non-HDL-C has been found to be a more consistent predictor of coronary heart disease (CHD) risk than LDL-C (Liu 2005, Robinson 2009).  Prior studies have shown that genetic variants that modify each of the components of non-HDL-C are associated with modification of cardiovascular disease risk, particularly incidence of CHD.

 

Ference and colleagues recently published a large-scale analysis of data from a group of 654,783 subjects, in 63 case-control or cohort studies, to investigate two sets of lipid-lowering genetic variants for the LDL receptor gene and the lipoprotein lipase (LPL) gene that predominantly affect LDL-C and TG, respectively (Ference 2019, Navar 2019).  The analysis included 91,129 cases of CHD.  Their investigation showed that both genetically-induced LDL-C reduction through the LDL receptor gene score and TG reduction through the LPL gene score were associated with significantly reduced CHD risk, findings which agree with those from prior investigations.

 

The authors then extended their analysis by investigating 168 genetic variants associated with either LDL-C or TG modification.  In order to make an “apples to apples” comparison, the associations were standardized to a 10 mg/dL difference in genetically-induced LDL-C reduction and a 50 mg/dL reduction in TG, which is equivalent to a 10 mg/dL reduction in VLDL-C (TG/5 = VLDL-C).  Each 10 mg/dL reduction in LDL-C was associated with 15.4% lower odds for CHD (odds ratio = 0.846) and each 10 mg/dL reduction in VLDL-C (50 mg/dL reduction in TG) was associated with 18.5% lower odds for CHD (odds ratio = 0.815).

 

Since LDL and VLDL particles each contain Apo B, the authors also investigated the effects of a 10 mg/dL genetically-induced reduction in Apo B.  The resulting odds ratio was 0.770, indicating 23% lower odds for CHD.  When 10 mg/dL reductions in all three (LDL-C, VLDL-C and Apo B) were included in the same model, only Apo B remained significant (odds ratio = 0.761). 

 

Comment on clinical implications.  The Apo B concentration represents the total number of circulating particles with atherogenic potential.  Most investigations have shown that Apo B predicts CHD risk slightly better than non-HDL-C, which is, in turn, a better predictor than LDL-C.  The present study extends those findings by showing that genetic modification of Apo B concentration is strongly associated with CHD risk, supporting a causal relationship.  The non-HDL-C concentration correlates strongly with the Apo B level because it represents cholesterol carried by the two main types of Apo B-containing lipoproteins, LDL and VLDL.

 

The National Lipid Association’s Recommendations for Patient-centered Management of Dyslipidemia identified non-HDL-C and LDL-C as co-primary targets of therapy for lipid modification (Jacobson 2014).  The recent American Heart Association/American College of Cardiology Guideline on the Management of Blood Cholesterol (Grundy 2018) also acknowledges the importance of non-HDL-C by identifying thresholds for either LDL-C or non-HDL-C for consideration of adding adjunctive therapy to a statin as a way of identifying patients who could potentially benefit from additional Apo B-containing lipoprotein reduction.  The results from this new study by Ference and colleagues suggest that a 10 mg/dL decline in VLDL-C has similar predictive value to that of a 10 mg/dL decline in LDL-C and that the predictive value of each is contained within the Apo B concentration.

 

In the US, Apo B measurement is not commonly completed.  Since non-HDL-C correlates strongly with the Apo B concentration, it can serve as a reasonable surrogate.  The National Lipid Association recommendations suggest goals for LDL-C of <70 mg/dL for those at very high risk and <100 mg/dL for others (Jacobson 2014).  The corresponding goals for non-HDL-C are <100 and <130 mg/dL, respectively.  It should be emphasized that the relationships show no evidence of thresholds, so reductions to levels of LDL-C and non-HDL-C well below 70 and 100 mg/dL, respectively, may be justified for some of the highest risk patients.  Such an approach is supported by results from studies of adjunctive therapies, including those with proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors and ezetimibe. 

 

Another important point with clinical relevance is that the reduction in CHD (or cardiovascular disease) risk with genetic variants is consistently larger than that observed in clinical trials of lipid-altering therapies.  For example, in a Cholesterol Treatment Trialists’ Collaboration analysis (2010), each mmol/L (38.7 mg/dL) reduction in LDL-C was associated with a 24% reduction in major CHD event risk, which means that a 10 mg/dL reduction induced by statin therapy would reduce CHD event risk by 6.8% (1 – 0.76(10/38.7) = 0.068 or 6.8%), considerably less than the 15.4% lower odds for CHD in the Ference investigation.  This likely reflects the greater length of time that individuals with genetic variants are exposed to altered levels of lipoproteins.  The implication is that even modestly lower levels of LDL-C and VLDL-C can have important impacts on CHD risk if maintained over an extended period, which highlights the importance of healthy diet and adequate physical activity.

 

The recently published results from the Reduction of Cardiovascular Events with EPA – Intervention Trial (REDUCE-IT) with eicosapentaenoic acid (EPA) ethyl esters showed an impressive reduction of 25% for major adverse cardiovascular events in patients with low baseline LDL-C (median 74 mg/dL) and elevated TG (median 216 mg/dL) (Bhatt 2019).  The placebo-corrected reductions in non-HDL-C and Apo B in the active treatment group were 10 mg/dL and 5-8 mg/dL, respectively.  The cardiovascular disease benefit was much larger than would be predicted based on the observed effects on non-HDL-C and Apo B over a period of ~5 years.  Similarly, studies of other TG-lowering drug therapies such as fibrates have shown substantial reductions in risk among subsets of patients with elevated TG, especially if accompanied by low HDL-C (Sacks 2010, Maki 2016).  In a meta-analysis completed by our group, cardiovascular disease risk reductions were 18% and 29% in studies of TG-lowering therapies in subgroups with elevated TG and elevated TG plus low HDL-C, respectively (Maki 2016).  It appears unlikely that these results can be explained entirely by changes in non-HDL-C or Apo B levels.  Therefore, additional research is needed to investigate pathways through which TG-lowering therapies affect cardiovascular risk.

 

In summary, genetically-induced reduction in LDL-C and VLDL-C (estimated as TG/5) are each associated with similar reductions in CHD risk.  The predictive value of each of these is contained within Apo B.  Since Apo B concentration is rarely measured in the US, non-HDL-C can serve as a surrogate marker and is a preferable target of therapy to LDL-C, because changes in both components of non-HDL-C (LDL-C and VLDL-C) appear to contribute similarly to risk alteration when compared on an “apples to apples” basis.  Thus, a 50 mg/dL reduction in TG (equivalent to a 10 mg/dL reduction in VLDL-C) should be expected to produce the same benefit for CHD risk as a 10 mg/dL lowering of LDL-C.

 

References

Bhatt DL, Steg PG, Miller M, et al. Cardiovascular risk reduction with icosapent ethyl for hypertriglyceridemia. N Engl J Med. 2019;380:11-22.

Cholesterol Treatment Trialists’ (CTT) Collaboration, Baigent C, Blackwell L, et al. Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials. Lancet. 2010;376:1670-1681.

Ference BA, Kastelein JJP, Ray KK, et al. Association of triglyceride-lowering LPL variants and LDL-C lowering LDLR variants with risk of coronary heart disease. JAMA. 2019;321:364-373.

Grundy SM, Stone NJ, Bailey AL, et al. 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA guideline on the management of blood cholesterol. Circulation. 2018 [Epub ahead of print].

Jacobson TA, Ito MK, Maki KC, et al. National Lipid Association recommendations for patient-centered management of dyslipidemia: part 1 – executive summary. J Clin Lipidol. 2014;8:473-488.

Liu J, Sempos C, Donahue RP, et al. Joint distribution of non-HDL and LDL cholesterol and coronary heart disease risk prediction among individuals with and without diabetes. Diabetes Care. 2005;28:1916-1921.

Maki KC, Guyton JR, Orringer CE, et al. Triglyceride-lowering therapies reduce cardiovascular disease event risk in subjects with hypertriglyceridemia. J Clin Lipidol. 2016;10:905-914.

Navar AM. The evolving story of triglycerides and coronary heart disease risk. JAMA. 321:347-349.

Robinson JG. Are you targeting non-high-density lipoprotein cholesterol? J Am Coll Cardiol. 2009;55:42-44.

Sacks FM, Carey VJ, Fruchart JC. Combination lipid therapy in type 2 diabetes. N Engl J Med. 2010;363:692-694.

Closeup of doctor checking patient daily report checklist

Meta-regression Characterizes the Relationships Between Changes in Dietary Cholesterol Intake and Plasma Low-density and High-density Lipoprotein Cholesterol Changes

Meta-regression Characterizes the Relationships Between Changes in Dietary Cholesterol Intake and Plasma Low-density and High-density Lipoprotein Cholesterol Changes

By Kevin C Maki, PhD and Orsolya M Palacios, PhD

 

Our group recently collaborated with scientists from the University of Cincinnati to conduct meta-regression analyses that investigated the dose-response relationships between changes in dietary cholesterol intake and changes in lipoprotein-cholesterol levels.  The results from these analyses were presented in June at the American Society for Nutrition’s annual Nutrition 2018 meetings,1,2 and have also recently been accepted for publication in the American Journal of Clinical Nutrition.This summary is also available in the Fall 2018 Newsletter of MB Clinical Research.

 

Elevated low-density lipoprotein cholesterol (LDL-C) is a major cardiovascular risk factor, and there is a strong inverse association between high-density lipoprotein cholesterol (HDL-C) concentration and cardiovascular risk.4  Dietary guidance generally recommends reducing intakes of saturated fatty acids (SFA) and trans fatty acids (TFA) to reduce LDL-C levels, but in recent years there has been a step back from making specific quantitative recommendations with regard to limiting dietary cholesterol intake.5,6  A key recommendation from the 2010 Dietary Guidelines was to limit consumption of dietary cholesterol to <300 mg/day, but this was not included in the 2015-2020 Dietary Guidelines, although they did explain that “this change does not suggest that dietary cholesterol is no longer important to consider when building healthy eating patterns.”6  The 2015 guidelines stated “Strong evidence from mostly prospective cohort studies but also randomized controlled trials has shown that eating patterns that include lower intake of dietary cholesterol are associated with reduced risk of CVD…”.6  However, the committee concluded that “More research is needed regarding the dose-response relationship between dietary cholesterol and blood cholesterol levels.  Adequate evidence is not available for a quantitative limit for dietary cholesterol specific to the Dietary Guidelines.” 6  The 2013 American Heart Association/American College of Cardiology Lifestyle Management Guideline also concluded “There is insufficient evidence to determine whether lowering dietary cholesterol reduces LDL-C.”5  One problem with the available evidence is that there are limited data examining how much of an impact on lipoprotein lipid levels is attributable to dietary cholesterol after controlling for intakes of dietary fatty acids (i.e., SFA, TFA, polyunsaturated fatty acids [PUFA] and monounsaturated fatty acids [MUFA]).

 

Therefore, the goal of these meta-regression analyses was to examine the impacts of changes in dietary cholesterol on lipoprotein cholesterol levels, after accounting for dietary fatty acids.1,2  This meta-regression examined results from 55 randomized controlled dietary intervention trials (n = 2652 subjects) using a Bayesian approach (with Markov chain Monte Carlo techniques) and adjustment for dietary fatty acids to determine the best fitting mathematical models to the data.7  No significant associations were observed between change in dietary cholesterol intake and change in triglyceride or very low density lipoprotein cholesterol level.

 

For LDL-C, the meta-regression results indicated a positive correlation using a linear model and two non-linear models (Michaelis-Menten and Hill models), even after accounting for intakes of SFA, PUFA, MUFA and, where possible, TFA.  The relationship was best characterized by the non-linear models across the full range of cholesterol changes (0-1500 mg/day).  A 100 mg/day dietary cholesterol change was predicted to be associated with an increase in LDL-C of ~ 4.5 mg/dL in LDL-C.  Baseline cholesterol intake was not a significant predictor of the LDL-C response to a change in dietary cholesterol.  The relationship between baseline LDL-C and LDL-C response was unclear, and needs further exploration.  For HDL-C, the meta-regression analyses did not indicate a clear relationship between the change in dietary cholesterol intake and the change in HDL-C levels when both men and women were included.  However, when analyzed according to sex, the linear model and the Michaelis-Menten non-linear model demonstrated an inverse relationship in men, and a positive relationship in women.  This suggests a possible interaction between sex and HDL-C response to dietary cholesterol.

 

Using the Mensink et al. equation, which is designed to calculate the effects of changes in carbohydrate and fatty acid intakes on serum lipid and lipoprotein levels, each 1% increase in SFA in exchange for carbohydrate is predicted to increase LDL-C by 1.23 mg/dL.8  These findings suggest that increasing dietary cholesterol by 100 mg/day or 200 mg/day would have effects comparable to increasing dietary SFA by 3.7% and 5.5%, respectively.

 

These results suggest that there is a clinically meaningful effect of dietary cholesterol on LDL-C concentration.  This finding from a pooled analysis of results from 55 studies aligns with those from the best-controlled individual studies, such as two published by Ginsberg et al.9,10  This dose-response analysis provides reference for clinicians and nutrition scientists on how changes in dietary cholesterol intake may impact plasma cholesterol levels, although considerable interindividual variability should be expected.9-11  The clinical implications of changes in HDL-C associated with increased dietary cholesterol intake remain uncertain.

 

References:

  1. Vincent MJ, Allen B, Maki KC, Palacios OM, Haber LT. Non-linear models best characterize the relationship between dietary cholesterol intake and circulating low-density lipoprotein cholesterol levels. Presented at American Society of Nutrition’s Nutrition 2018 meetings, June 9-12, 2018, Boston MA.
  2. Palacios OM, Vincent MJ, Allen B, Haber LT, Maki KC. The effect of dietary cholesterol on high-density lipoprotein cholesterol levels in men and women: a meta-analysis of randomized controlled trials. Presented at American Society of Nutrition’s Nutrition 2018 meetings, June 9-12, 2018, Boston MA.
  3. Vincent MJ, Allen B, Palacios OM, Haber LT, Maki KC. Meta-regression analysis of the effects of dietary cholesterol intake on low- and high-density lipoprotein cholesterol. Am J Clin Nutr. 2018; In Press.
  4. Jacobson TA, Ito MK, Maki KC, Orringer CE, Bays HE, Jones PH, McKenney JM, Grundy SM, Gill EA, Wild RA, Wilson DP, Brown WV. National Lipid Association recommendations for patient-centered management of dyslipidemia: part 1 – executive summary. J Clin Lipidol. 2014;8:473-488.
  5. Eckel RH, Jakicic JM, Ard JD, de Jesus JM, Houston Miller N, Hubbard VS, Lee IM, Lichtenstein AH, Loria CM, Millen BE, Nonas CA, Sacks FM, Smith SC Jr., Svetkey LP, Wadden TA, Yanovski SZ; American College of Cardiology/American Heart Association Task Force on Practice Guidelines. 2013 AHA/ACC guideline on lifestyle managemnet to reduce cardiovascular risk: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2014;63(25 Pt B):2960-2984.
  6. U.S. Department of Health and Human Services and U.S Department of Agriculture. 2015-2020 Dietary Guidelines for Americans. 8th edition. December 2015. Available at https://health.gov/dietaryguidelines/2015/resources/2015-2020_Dietary_Guidelines.pdf.
  7. The Stan Development Team. RStan: the R interface to Stan. R package version 2.16.2. 2017. Available at http://mc-stan.org.
  8. Mensink RP, Zock PL, Kester AD, Katan MB. Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials. Am J Clin Nutr. 2003;77:1146-1155.
  9. Ginsberg HN, Karmally W, Siddiqui M, Holleran S, Tall AR, Rumsey SC, Deckelbaum RJ, Blaner WS, Ramakrishnan R. A dose-response study of the effects of dietary cholesterol on fasting and postprandial lipid and lipoprotein metabolism in healthy young men. Arterioscler Thromb. 1994;14:576-586.
  10. Ginsberg HN, Karmally W, Siddiqui M, Holleran S, Tall AR, Blaner WS, Ramakrishnan R. Increases in dietary cholesterol are associated with modest increases in both LDL and HDL cholesterol in healthy young women. Arterioscler Thromb Vasc Biol. 1995;15:169- 178.
  11. Jacobson TA, Maki KC, Orringer CE, Jones PH, Kris-Etherton P, Sikand G, La Forge R, Daniels SR, Wilson DP, Morris PB, Wild RA, Grundy SM, Daviglus M, Ferdinand KC, Vijayaraghavan K, Deedwania PC, Aberg JA, Liao KP, McKenney JM, Ross JL, Braun LT, Ito MK, Bays HE, Brown WV, Underberg JA, NLA Expert Panel. National Lipid Association recommendations for patient-centered management of dyslipidemia: Part 2. J Clin Lipidol. 2015;9(6 Suppl):S1-S122.
Photo by Kelly Sikkema

ODYSSEY Outcomes Trial: Topline Results and Clinical Implications

ODYSSEY Outcomes Trial: Topline Results and Clinical Implications

By Kevin C Maki, PhD, CLS, FNLA; Kristen N Smith, PhD, RD, LD; Mary R Dicklin, PhD

 Background:

Cardiovascular disease (CVD) event risk is high in those with recent acute coronary syndromes (ACS), despite treatment with evidence-based preventive therapies. Prior research has shown that CVD event risk is lowered when low-density lipoprotein cholesterol (LDL-C) is lowered through various means, such as:

  • Statin therapy (compared with placebo)2
  • High-intensity statin therapy (compared with moderate-intensity statin therapy)3
  • Ezetimibe added to statin therapy (compared with placebo)4
  • Anacetrapib added to statin therapy (compared with placebo)5
  • Evolocumab added to statin therapy (compared with placebo)6

Alirocumab is a fully human monoclonal antibody against proprotein convertase subtilisin kexin type 9 (PCSK9), a validated target for risk reduction in patients with stable atherosclerotic CVD.7-8. Research outcomes on alirocumab show that it reduces LDL-C (sustained reductions) and other atherogenic lipoproteins7 with documented safety and tolerability.8

The hypothesis of the Evaluation of Cardiovascular Outcomes after an Acute Coronary Syndrome During Treatment with Alirocumab (ODYSSEY Outcomes) trial was that alirocumab, versus placebo, reduces cardiovascular (CV) morbidity and mortality after recent ACS in patients with elevated levels of atherogenic lipoproteins despite intensive or maximum-tolerated statin therapy.9

 Methods:

This study was a randomized, double-blind, placebo-controlled, parallel group study of 18,924 patients randomized at 1315 sites in 57 countries between November 2, 2012 and November 11, 2017.1,9

Key inclusion criteria:

  • Age ≥40 years
  • ACS
    • 1 to 12 months prior to randomization [acute myocardial infarction (MI) or unstable angina]
  • High-intensity statin therapy
    • Atorvastatin 40 to 80 mg daily, or
    • Rosuvastatin 20 to 40 mg daily, or
    • Maximum tolerated dose of one of these agents for ≥2 weeks
    • Patients not taking statins were authorized to participate if tolerability issues were present and documented
  • Inadequate control of lipids
    • LDL-C ≥70 mg/dL (1.8 mmol/L), or
    • Non-high-density lipoprotein cholesterol (non-HDL-C) ≥100 mg/dL (2.6 mmol/L), or
    • Apolipoprotein B ≥80 mg/dL

 

Key exclusion criteria:

  • Uncontrolled hypertension
  • NYHA class III or IV heart failure; left ventricular ejection fraction <25% if measured
  • History of hemorrhagic stroke
  • Fasting triglycerides >400 mg/dL (4.52 mmol/L)
  • Use of fibrates, other than fenofibrate or fenofibric acid
  • Recurrent ACS within 2 weeks prior to randomization
  • Coronary revascularization performed within 2 weeks prior to or after randomization
  • Liver transaminases >3 x upper limit of normal; hepatitis B or C infection
  • Creatine kinase >3 x upper limit of normal
  • Estimated glomerular filtration rate <30 mL/min/1.73 m2
  • Positive pregnancy test

 

Primary Efficacy Outcome

Major Secondary Efficacy Endpoints

Other Secondary and Safety Endpoints

Time of first occurrence:

§  Coronary heart disease (CHD) death, or

§  Non-fatal MI, or

§  Fatal or non-fatal ischemic stroke, or

§  Unstable angina requiring hospitalization

Tested in the following hierarchical sequence:

§  CHD event: CHD death, non-fatal MI, unstable angina requiring hospitalization, or ischemia-driven coronary revascularization

§  Major CHD event: CHD death or non-fatal MI

§  CV event: CV death, non-fatal CHD event, or non-fatal ischemic stroke

§  All-cause death, non-fatal MI, non-fatal ischemic stroke

§  CHD death

§  CV death

§  All-cause death

Secondary endpoints:

§  Components of the primary endpoint considered individually:

·       CHD death

·       Non-fatal MI

·       Fatal and non-fatal ischemic stroke

·       Unstable angina requiring hospitalization

§  Ischemia-driven coronary revascularization

§  Congestive heart failure requiring hospitalization

 

Safety endpoints:

§  Adverse events

§  Laboratory assessments

Patients screened for this study completed a run-in period of 2 to 16 weeks on high-intensity or maximum-tolerated dose of atorvastatin or rosuvastatin. If at least one lipid entry criterion was met, then the subject was randomized to receive either subcutaneous alirocumab (75 or 150 mg) or placebo every 2 weeks. In order to maximize the number of patients in the target LDL-C range (25-50 mg/dL), alirocumab was blindly titrated or subjects were blindly switched to placebo if they were either substantially above or below (<15 mg/dL for LDL-C) the target range.

Results:

Of the 18,924 patients randomized for this study:

  • 9462 were assigned to alirocumab and 9462 received placebo
  • Median follow-up was 2.8 years (interquartile range limits 2.3-3.4 years)
  • 8242 (44%) patients with potential follow-up ≥3 years
  • 1955 patients experienced a primary endpoint; 726 patients died

Topline results showed that treatment with alirocumab was associated with significant reductions in LDL-C, and these reductions remained consistent over time.  Mean baseline and on-treatment LDL-C values are shown in the table below.

 

 

Placebo

(n = 9462)

Alirocumab

(n = 9462)

Baseline

  87.0 mg/dL

87.0 mg/dL

4 months

  93.3 mg/dL

39.8 mg/dL

12 months

  96.4 mg/dL

48.0 mg/dL

48 months

101.4 mg/dL

66.4 mg/dL

The “on-treatment” analysis showed that mean LDL-C was lowered by 55.7 mg/dL (-62.7%) in the alirocumab group vs. placebo at 4 months, 54.1 mg/dL (-61.0%) at 12 months and 48.1 mg/dL (-54.7%) at 48 months.

Several endpoints were significantly less frequent in the alirocumab group vs. placebo. Major adverse cardiac events (MACE; includes CHD death, non-fatal MI, ischemic stroke, or unstable angina requiring hospitalization) are shown in the table below, along with other endpoints.

 

Endpoint

Alirocumab

(n = 9462)

n (%)

Placebo

(n = 9462)

n (%)

Hazard Ratio (95% Confidence Interval)

Log-rank

P-value

MACE

903 (9.5)

1052 (11.1)

0.85 (0.78, 0.93)

0.0003

     CHD death

205 (2.2)

222 (2.3)

0.92 (0.76, 1.11)

0.38

     Non-fatal MI

626 (6.6)

722 (7.6)

0.86 (0.77, 0.96)

0.006

     Ischemic stroke

111 (1.2)

152 (1.6)

0.73 (0.57, 0.93)

0.01

     Unstable angina

37 (0.4)

60 (0.6)

0.61 (0.41, 0.92)

0.02

Secondary

       

     CHD event

1199 (12.7)

1349 (14.3)

0.88 (0.81, 0.95)

0.001

     Major CHD

     event

793 (8.4)

899 (9.5)

0.88 (0.80, 0.96)

0.006

     CV event

1301 (13.7)

1474 (15.6)

0.87 (0.81, 0.94)

0.0003

     Death, MI,

     ischemic stroke

973 (10.3)

1126 (11.9)

0.86 (0.79, 0.93)

0.0003

     CHD death

205 (2.2)

222 (2.3)

0.92 (0.76, 1.11)

0.38

     CV death

240 (2.5)

271 (2.9)

0.88 (0.74, 1.05)

0.25

     All-cause death

334 (3.5)

392 (4.1)

0.85 (0.73, 0.98)

0.026*

(nominal)

Several pre-specified subgroup analyses for the primary outcome variable were presented, including, notably, an analysis by baseline LDL-C categories of <80, 80-99, and ≥100 mg/dL.  Although the test for heterogeneity of response across subgroups was not statistically significant (p = 0.09), the hazard ratio (HR) for the comparison of alirocumab to placebo was numerically lower for the subgroup with baseline LDL-C ≥100 mg/dL [HR 0.76, 95% confidence interval (CI) 0.65 to 0.87] than for those with baseline LDL-C <80 mg/dL (HR 0.86, 95% CI 0.74 to 1.01) or 80-99 mg/dL (HR 0.96, 95% CI 0.92 to 1.14).

Comment by Kevin C Maki, PhD, CLS, FNLA:

When compared with placebo, the use of alirocumab 75 or 150 mg every two weeks, aiming for LDL-C levels of 25-50 mg/dL (and allowing levels as low as 15 mg/dL), led to reduced MACE, MI and ischemic stroke, and was associated with reduced all-cause death. Treatment was safe and well tolerated. CV and CHD death were not significantly reduced. Therefore, the results for total mortality should be viewed with caution, since roughly 70% of total mortality was attributable to CV causes. 

Subgroup analyses identified numerically larger benefits for the primary outcome in subjects with baseline levels of LDL-C ≥100 mg/dL (median LDL-C 118 mg/dL). However, the test for heterogeneity of effect across subgroups was not statistically significant (p = 0.09).  The proportional risk reductions for subjects with baseline LDL-C <80, 80-99 and ≥100 mg/dL were 14%, 4% and 24%, respectively. Only the subgroup with LDL-C ≥100 mg/dL showed a statistically significant reduction in the alirocumab group compared with placebo. That was also true for all-cause mortality, which was reduced by 29% in the alirocumab group vs. placebo in subjects with baseline LDL-C ≥100 mg/dL, but was not significantly reduced in the other subgroups. This finding should also be interpreted with caution because the test for heterogeneity was, again, not significant (p = 0.12). 

The US Institute for Clinical and Economic Review (ICER) provided new estimates of the price range that would be acceptable for the drug, based on the results of the ODYSSEY Outcomes trial.10 ICER calculated two updated value-based price benchmarks, net of rebates and discounts, for alirocumab in patients with a recent acute coronary event:  $2300-$3400 per year if used to treat all patients who meet trial eligibility criteria, and $4500-$8000 per year if used to treat higher-risk patients with LDL-C ≥100 mg/dL despite intensive statin therapy. The manufacturers of alirocumab (Sanofi and Regeneron Pharmaceuticals, Inc.) have announced plans for a reduced price for the drug that will be in the range of the $4500-$8000 identified by ICER, which is substantially below the “list price” of approximately $14,000 per year that had been charged initially.

A number of commentaries from experts in the field have suggested that this class of medications is appropriate for use mainly in patients in whom LDL-C is ≥100 mg/dL, based on the results from the analyses presented at the American College of Cardiology meeting (and not yet published in a peer reviewed journal), including the cost-effectiveness evaluation by ICER. For example, the highly respected cardiologist Milton Packer, MD wrote a piece in which he stated:11

“…the benefit in the entire trial was driven entirely by the effect seen in 5,629 patients who started with LDL cholesterol >100 mg/dL. There was no benefit in patients with lower values for baseline LDL cholesterol.”

With all due respect to Dr. Packer and others who hold this opinion, I view this conclusion as premature for several reasons. The test for heterogeneity (treatment by subgroup interaction) across baseline LDL-C categories was not significant at an alpha of 5%, showing a p-value of 0.09.  Moreover, the study was not designed with sufficient statistical power to reliably differentiate effects across baseline LDL-C categories. This lack of statistical power for tests of heterogeneity of treatment effects argues for caution in the clinical application of such findings, even when the test for heterogeneity is pre-specified and/or when it does reach statistical significance.

Sir Richard Peto, the eminent biostatistician and epidemiologist from the University of Oxford has quipped that only one thing is worse than doing subgroup analyses for a clinical trial, and that is believing the results! To demonstrate the potential unreliability, Peto reported on a set of subgroup analyses from the Second International Study of Infarct Survival (ISIS-2).12  In the trial overall, the survival advantage produced by aspirin for patients with suspected myocardial infarction was 23%, which was highly statistically significant (p < 0.000001).13 ISIS-2 patients were divided into 12 subgroups according to their astrological sign, and the treatment effect of aspirin compared with placebo was calculated in each subgroup. The results ranged from no apparent effect of aspirin in two subgroups (Libra and Gemini) to aspirin being associated with a halving of the mortality in another (Capricorn).

Results from subgroup analyses are useful for generating hypotheses to test prospectively, but should not, in most cases, be applied as the sole basis for clinical practice decisions without replication in other trials, and, ideally, prospective testing in one or more trials designed for the purpose of evaluating possible differences across subgroups in clinical response.  Regarding the results for the subgroup with baseline LDL-C <100 mg/dL in ODYSSEY Outcomes, it should be noted that the authors of the paper from the Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk (FOURIER) trial with the other approved PCSK9 inhibitor agent, evolocumab, report the following:6

“The benefits were also consistent across quartiles of baseline LDL cholesterol levels, from patients in the top quartile, who had a median LDL cholesterol level of 126 mg per deciliter (interquartile range, 116 to 143) (3.3 mmol per liter [interquartile range, 3.0 to 3.7]) at baseline, down to those in the lowest quartile, who had a median LDL cholesterol level of 74 mg per deciliter (interquartile range, 69 to 77) (1.9 mmol per liter [interquartile range, 1.8 to 2.0]) at baseline.”

Results from other trials such as the Improved Reduction of Outcomes: Vytorin Efficacy International Trial4 (IMPROVE-IT; statin plus ezetimibe) and the HPS3/TIMI55 Randomized Evaluation of the Effects of Anacetrapib through Lipid-modification5 (REVEAL; statin plus anacetrapib) have shown benefits with atherogenic cholesterol lowering that are generally consistent with the results observed in statin trials based on the Cholesterol Treatment Trialists’ (CTT) analyses,14 i.e., a HR of 0.78 (22% reduction) per mmol/L (38.7 mg/dL) reduction in LDL-C, despite average baseline LDL-C levels below 100 mg/dL (~94 mg/dL in IMPROVE-IT and 61 mg/dL in HPS2/TIMI55-REVEAL).  Notably, in both IMPROVE-IT and HPS3/TIMI55-REVEAL, the placebo and active treatment group Kaplan-Meier curves did not clearly separate for the first 2.0 to 2.5 years. In a prior study with evacetrapib [Assessment of Clinical Effects of Cholesteryl Ester Transfer Protein Inhibition with Evacetrapib in Patients at a High Risk for Vascular Outcomes (ACCELERATE)], no CVD event benefit (or harm) was observed over a median follow-up period of 2.2 years, despite modest lowering of LDL-C.15  The median follow-up period for both of the PCSK9 inhibitor trials (FOURIER, 2.2 years and ODYSSEY Outcomes, 2.8 years) was short compared with those from most trials of statins, which had median follow-up periods that averaged roughly 5 years.14 In fact, the FOURIER investigators reported that:

“… in FOURIER, the magnitude of the risk reduction with regard to the key secondary end point appeared to grow over time, from 16% during the first year to 25% beyond 12 months, which suggests that the translation of reductions in LDL cholesterol levels into cardiovascular clinical benefit requires time.”

Findings from studies of genetic variants that alter atherogenic cholesterol levels suggest that the benefits of maintaining lower levels may not be fully apparent after only a few years of intervention. The prototypical example of this is one of the findings that led to the development of the PCSK9 inhibitor class of lipid-altering agents. Cohen et al.16 reported that a nonsense loss-of-function mutation in the PCSK9 gene was associated with a 38 mg/dL (0.98 mmol/L) lower average level of LDL-C, and a missense mutation was associated with a 21 mg/dL (0.54 mmol/L) reduction in LDL-C. Based on the CTT relationship, the predicted reductions in CVD event risk would have been roughly 22% and 13%, respectively. However, the observed reductions in CVD (CHD and stroke) incidence were approximately 50% and 37%, respectively (estimated from data presented in the paper). The reductions in risk were most evident for CHD, where HRs were 0.11 (89% reduction) and 0.50 (50% reduction) for those with the nonsense and missense mutations, respectively. These results, and those from many other studies of lipid-altering genetic variants, suggest a greater CVD event risk reduction than would be predicted from the effects of statin and other lipid-altering therapies on risk.17 A likely explanation is that genetic variants produce differences that are maintained over decades, rather just a few years duration, as is the case in randomized, controlled intervention trials. 

Thus, there are reasons to believe that “lower is probably better” for atherogenic cholesterol levels with regard to CVD event reduction in high-risk patients, even if the baseline level of LDL-C is less than 100 mg/dL. Atherosclerosis is a disease that develops and progresses over decades. Thus, it seems possible, and indeed, likely, that benefits will be observed with therapy to further reduce atherogenic cholesterol among those with LDL-C less than 100 mg/dL over follow-up periods longer than the 2- to 3-year median durations in the ODYSSEY Outcomes and FOURIER trials. At present, this is a hypothesis that remains to be verified with additional clinical research. Given that the subgroup with baseline LDL-C <100 mg/dL who received placebo in the ODYSSEY Outcomes trial experienced an event rate above 3% per year, substantial residual risk is present in such patients. Dr. Packer ended his commentary by saying “The ODYSSEY trial shows that we may have reached the limits of what we can achieve by lowering lipids.” My view is that the potential for aggressive atherogenic cholesterol reduction to lower CVD event risk in those with recent ACS (and other high-risk patients) has not been fully evaluated. Accordingly, efforts to understand the effects of atherogenic cholesterol lowering in high-risk patients with LDL-C levels <100 mg/dL should remain an important priority.

References:

  1. Schwartz GG, Szarek M, Bhatt DL, et al. The ODYSSEY OUTCOMES trial: topline results. Alirocumab in patients after acute coronary syndrome. Presented at ACC.18 67th Annual Scientific Session & Expo. Acc.18 Joint ACC/JACC Late-breaking clinical trials. Accessed at https://accscientificsession.acc.org/features/2018/03/video-sanofi-regeneron on March 15, 2018.
  2. Schwartz GG, Olsson AG, Ezekowitz MD, et al. Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial. JAMA. 2001;285(13):1711-1718.
  3. Cannon CP, Braunwald E, McCabe CH, et al. Intensive versus moderate lipid lowering with statins after acute coronary syndromes. N Engl J Med. 2004;350(15):1495-1504.
  4. Cannon CP, Blazing MA, Giugliano RP, et al. Ezetimibe added to statin therapy after acute coronary syndromes. N Engl J Med. 2015;372(25):2387-2397.
  5. HPS3/TIMI55-REVEAL Collaborative Group, Bowman L, Hopewell JC, et al. Effects of anacetrapib in patients with atherosclerotic vascular disease. N Engl J Med. 2017;377(13):1217-1227.
  6. Sabatine MS, Giugliano RP, Keech AC, et al. Evolocumab and clinical outcomes in patients with cardiovascular disease. N Engl J Med. 2017;376(18):1713-1722.
  7. Robinson JG, Farnier M, Krempf M, et al. Efficacy and safety of alirocumab in reducing lipids and cardiovascular events. N Engl J Med. 2015;372(16):1489-1499.
  8. Robinson JG, Rosenson RS, Farnier M, et al. Safety of very low low-density lipoprotein cholesterol levels with alirocumab: pooled data from randomized trials. J Am Coll Cardiol. 2017;69(5):471-482.
  9. Schwartz GG, Bessac L, Berdan LG, et al. Effect of alirocumab, a monoclonal antibody to PCSK9, on long-term cardiovascular outcomes following acute coronary syndromes: rationale and design of the ODYSSEY outcomes trial. Am Heart J. 2014;168(5):682-689.
  10. Institute for Clinical and Economic Review, 2018. Alirocumab for treatment of high cholesterol: effectiveness and value. Preliminary New Evidence Update. March 10, 2018. Accessed at https://icer-review.org/wp-content/uploads/2018/03/Alirocumab-Preliminary-New-Evidence-Update_03102018.pdf on March 23, 2018.
  11. Packer, M. Confessions and omens from the ODYSSEY Trial - Milton Packer assesses his predictions in the future of lipid research. MEDPAGE TODAY, March 14, 2018. Accessed at https://www.medpagetoday.com/blogs/revolutionandrevelation/71755 on March 23, 2018.
  12. Peto R. Current misconception 3: that subgroup-specific trial mortality results often provide a good basis for individualising patient care. Br J Cancer. 2011;104:1057-1058.
  13. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988;2(8607):349-360.
  14. Baigent C, Keech A, Kearney PM, et al. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. Lancet. 2005;366(9493):1267-1278.
  15. Lincoff AM, Nicholls SJ, Riesmeyer JS, et al. Evacetrapib and cardiovascular outcomes in high-risk vascular disease. N Engl J Med. 2017;376(20):1933-1942.
  16. Cohen JC, Boerwinkle E, Mosley TH, Jr., et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med. 2006;354(12):1264-1272.

17.       Ference BA. Mendelian randomization studies: using naturally randomized genetic data to fill evidence gaps. Curr Opin

An Independent Summary of Low-Density Lipoprotein Cholesterol Lowering With Evolocumab and Outcomes in Patients with Peripheral Artery Disease: Insights from the FOURIER Trial

An Independent Summary of Low-Density Lipoprotein Cholesterol Lowering With Evolocumab and Outcomes in Patients with Peripheral Artery Disease: Insights from the FOURIER Trial (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk).1

An Independent Summary of Low-Density Lipoprotein Cholesterol Lowering With Evolocumab and Outcomes in Patients with Peripheral Artery Disease: Insights from the FOURIER Trial (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk).1

By Kristen N Smith, PhD, RD, LD; Kevin C Maki, PhD

Background: Peripheral artery disease (PAD) is considered an indicator of a malignant vascular phenotype with event occurrences greater than other stable populations with atherosclerosis.2,3 Patients who have symptomatic PAD have a greater risk of major adverse cardiovascular events (MACE)4 and they also experience significant morbidity from major adverse limb events (MALE).5,6

 

Condition

Event Includes

Major adverse cardiovascular events (MACE)

Myocardial infarction (MI), stroke and cardiovascular death

Major adverse limb events (MALE)

Acute limb ischemia, urgent peripheral revascularization and major amputation

 

Methods of lowering lipid levels have been shown to help reduce MACE in stable patients with coronary heart disease or atherosclerosis risk factors, but there have been few well-developed, well-powered prospective randomized trials of low-density lipoprotein (LDL-C) reduction in patients with PAD.7 In particular, these prior studies have not examined relationships between the ability of LDL-C reductions to consequently reduce the risk of MALE.5,8-10  There is a shortage of information regarding the impact of reducing LDL-C on event risk in patients with PAD without prior MI or stroke.7,11,12

 In the FOURIER (Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk) trial, researchers gathered data on the use of evolocumab (a monoclonal antibody that inhibits proprotein convertase subtilisin kexin type 9 [PCSK9] and lowers LDL-C levels) compared with placebo in statin-treated patients with atherosclerotic disease in the coronary, cerebrovascular or peripheral artery bed. The FOURIER trial allowed the testing of the following hypotheses:1

  1. Patients with PAD would be at greater risk of MACE relative to patients with coronary or cerebrovascular disease without PAD;
  2. Consistent relative risk reductions in MACE with evolocumab would translate to larger absolute risk reductions in patients with PAD relative to those without;
  3. LDL-C reduction with evolocumab would significantly reduce MALE with benefits extending to very low levels of LDL-C.

This secondary analysis of the FOURIER study provided additional information on patients not only with cardiovascular risk factors, but also specifically of those meeting the criteria for PAD. 

 Methods: The FOURIER study was a randomized, double-blind, placebo-controlled, multinational clinical trial (1242 sites in 49 countries) investigating evolocumab versus placebo in 27,564 subjects with atherosclerotic disease. These subjects were followed for a median of 2.2 years.

 Inclusion Criteria:

  • Subjects were 40 to 85 years of age;
  • Clinically evident atherosclerotic cardiovascular disease (history of MI, non-hemorrhagic stroke or symptomatic PAD)
  • Fasting LDL-C of 70 mg/dL or greater OR non-high-density-lipoprotein cholesterol of 100 mg/dL or higher while on an optimized regimen of lipid-lowering medication (“preferably a high-intensity statin but must have been at least atorvastatin at a dose of 20 mg daily or its equivalent, with or without ezetimibe”).

 For the subset of patients with PAD, qualifications were met if the subjects had intermittent lower extremity claudication and an ankle brachial index <0.85, or a history of peripheral artery revascularization procedure or a history of amputation attributable to atherosclerotic disease.

Study endpoints are outlined in the following table: 

Primary End Points

Secondary End Points

Additional End Points of Interest

Major cardiovascular events, the composite of:

 

§  Cardiovascular death

§  MI

§  Stroke

§  Hospitalization for unstable angina

§  Coronary revascularization

The composite of:

 

§  Cardiovascular death

§  MI

§  Stroke

 

MALE:

 

§  Acute limb ischemia

§  Major amputation

§  Urgent peripheral revascularization for ischemia

 

 

Patients in this study were randomly assigned in a 1:1 ratio to receive either subcutaneous injections of evolocumab (either 140 mg every 2 weeks or 420 mg every month, according to patient preference) or matching placebo.

Results: Of the 27,564 subjects participating in the FOURIER study, 3642 patients (13.2%) had PAD. Within this subset, 1505 (41%) met the criteria for PAD but had no prior MI or stroke.  Use of evolocumab significantly reduced the primary end point (outlined above) in patients with PAD (hazard ratio [HR] 0.79; 95% confidence interval [CI] 0.66-0.94; p=0.0098) and in patients without PAD (HR 0.86; 95% CI 0.80-0.93; p=0.0003; pinteraction=0.40). Results of the secondary end point (outlined above) mirrored the primary end point. The HR for patients with PAD was 0.73 (95% CI 0.59-0.91; p=0.0040) and was 0.81 (95% CI 0.73-0.90; p<0.0001) for those without PAD (pinteraction=0.41). Since patients with PAD experience elevated risk, they also had larger absolute risk reductions for the primary end point (3.5% with PAD, 1.6% without PAD) and the secondary end point (3.5% for patients with PAD, 1.4% for those without PAD).

Treatment with evolocumab was also associated with reduced risk of MALE in all patients (HR 0.58; 95% CI, 0.38-0.88; p=0.0093) and these effects were consistent regardless of PAD status. The researchers noted a consistent relationship between lower (achieved) LDL-C and reduced risk of limb events (p=0.026 for the beta coefficient) that extended to values <10 mg/dL.

There were no differences in the incidence of adverse events or serious adverse events with evolocumab relative to placebo. Rates of treatment discontinuation were also similar between groups (1.3% evolocumab versus 1.5% placebo, p=0.57).

Comment: The FOURIER study provided evidence showing that patients with PAD were at greater risk of both MACE and MALE compared to patients with prior MI or stroke and no PAD, and captured the effects of the use of evolocumab as an adjunct to statin. Ultimately, the use of evolocumab assisted in reducing the risk of MACE (greater reductions in the subjects with PAD) and the concomitant LDL-C reductions associated with evolocumab treatment also decreased risk of MALE.

The FOURIER study provided clinical outcomes in a broad population with polyvascular disease and within those with isolated PAD (no history of MI or stroke). The results illustrated the benefits of the use of evolocumab added as an adjunct to a statin treatment in both groups, with minimal adverse events. Based on the results of this study, the combined usage of evolocumab plus statin treatment provided significant reductions in cardiovascular disease-related outcomes and warrants additional attention as a method of improving health outcomes in patients with PAD.

References:

  1. Bonaca MP, Nault P, Giugliano RP, et al. Low-Density Lipoprotein Cholesterol Lowering With Evolocumab and Outcomes in Patients With Peripheral Artery Disease: Insights From the FOURIER Trial (Further Cardiovascular Outcomes Research With PCSK9 Inhibition in Subjects With Elevated Risk). Circulation. 2017.
  2. Criqui MH, Aboyans V. Epidemiology of peripheral artery disease. Circulation research. 2015;116(9):1509-1526.
  3. Bonaca MP, Bhatt DL, Storey RF, et al. Ticagrelor for Prevention of Ischemic Events After Myocardial Infarction in Patients With Peripheral Artery Disease. Journal of the American College of Cardiology. 2016;67(23):2719-2728.
  4. Aboyans V, Ricco JB, Bartelink MEL, et al. 2017 ESC Guidelines on the Diagnosis and Treatment of Peripheral Arterial Diseases, in collaboration with the European Society for Vascular Surgery (ESVS): Document covering atherosclerotic disease of extracranial carotid and vertebral, mesenteric, renal, upper and lower extremity arteriesEndorsed by: the European Stroke Organization (ESO)The Task Force for the Diagnosis and Treatment of Peripheral Arterial Diseases of the European Society of Cardiology (ESC) and of the European Society for Vascular Surgery (ESVS). European heart journal. 2017.
  5. Kumbhani DJ, Steg PG, Cannon CP, et al. Statin therapy and long-term adverse limb outcomes in patients with peripheral artery disease: insights from the REACH registry. European heart journal. 2014;35(41):2864-2872.
  6. Jones WS, Baumgartner I, Hiatt WR, et al. Ticagrelor Compared With Clopidogrel in Patients With Prior Lower Extremity Revascularization for Peripheral Artery Disease. Circulation. 2017;135(3):241-250.
  7. Aung PP, Maxwell HG, Jepson RG, Price JF, Leng GC. Lipid-lowering for peripheral arterial disease of the lower limb. The Cochrane database of systematic reviews. 2007(4):CD000123.
  8. Aronow WS, Nayak D, Woodworth S, Ahn C. Effect of simvastatin versus placebo on treadmill exercise time until the onset of intermittent claudication in older patients with peripheral arterial disease at six months and at one year after treatment. The American journal of cardiology. 2003;92(6):711-712.
  9. Mohler ER, 3rd, Hiatt WR, Creager MA. Cholesterol reduction with atorvastatin improves walking distance in patients with peripheral arterial disease. Circulation. 2003;108(12):1481-1486.
  10. Spring S, Simon R, van der Loo B, et al. High-dose atorvastatin in peripheral arterial disease (PAD): effect on endothelial function, intima-media-thickness and local progression of PAD. An open randomized controlled pilot trial. Thrombosis and haemostasis. 2008;99(1):182-189.
  11. Bonaca MP, Scirica BM, Creager MA, et al. Vorapaxar in patients with peripheral artery disease: results from TRA2{degrees}P-TIMI 50. Circulation. 2013;127(14):1522-1529, 1529e1521-1526.
  12. Hiatt WR, Fowkes FG, Heizer G, et al. Ticagrelor versus Clopidogrel in Symptomatic Peripheral Artery Disease. The New England journal of medicine. 2017;376(1):32-40.

 

test tubes