Better biomarkers are urgently needed to improve diagnosis, guide molecularly targeted therapy and monitor activity and therapeutic response across a wide spectrum of disease. Proteomics methods based on mass spectrometry hold special promise for the discovery of novel biomarkers that might form the foundation for new clinical blood tests, but to date their contribution to the diagnostic armamentarium has been disappointing. This is due in part to the lack of a coherent pipeline connecting marker discovery with well-established methods for validation. Advances in methods and technology now enable construction of a comprehensive biomarker pipeline from six essential process components: candidate discovery, qualification, verification, research assay optimization, biomarker validation and commercialization. Better understanding of the overall process of biomarker discovery and validation and of the challenges and strategies inherent in each phase should improve experimental study design, in turn increasing the efficiency of biomarker development and facilitating the delivery and deployment of novel clinical tests.

Protein biomarker discovery and validation: the long and uncertain path to clinical utility.

Background—Elevated plasma concentrations of C-reactive protein (CRP) are associated with increased cardiovascular risk. We evaluated whether long-term therapy with pravastatin, an agent that reduces cardiovascular risk, might alter levels of this inflammatory parameter. Methods and Results—CRP levels were measured at baseline and at 5 years in 472 randomly selected participants in the Cholesterol and Recurrent Events (CARE) trial who remained free of recurrent coronary events during follow-up. Overall, CRP levels at baseline and at 5 years were highly correlated (r50.60, P,0.001). However, among those allocated to placebo, median CRP levels and the mean change in CRP tended to increase over time (median change, 14.2%; P50.2 and mean change, 10.07 mg/dL; P50.04). By contrast, median CRP levels and the mean change in CRP decreased over time among those allocated to pravastatin (median change, 217.4%; P50.004 and mean change, 20.07 mg/dL; P50.002). Thus, statistically significant differences were observed at 5 years between the pravastatin and placebo groups in terms of median CRP levels (difference, 221.6%; P50.007), mean CRP levels (difference, 237.8%; P50.002), and absolute mean change in CRP (difference, 20.137 mg/dL; P50.003). These effects persisted in analyses stratified by age, body mass index, smoking status, blood pressure, and baseline lipid levels. Attempts to relate the magnitude of change in CRP to the magnitude of change in lipids in both the pravastatin and placebo groups did not reveal any obvious relationships. Conclusions—Among survivors of myocardial infarction on standard therapy plus placebo, CRP levels tended to increase over 5 years of follow-up. In contrast, randomization to pravastatin resulted in significant reductions in this inflammatory marker that were not related to the magnitude of lipid alterations observed. Thus, these data further support the potential for nonlipid effects of this agent. (Circulation. 1999;100:230-235.)

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Long-Term Effects of Pravastatin on Plasma Concentration of C-reactive Protein

Background —We studied whether inflammation after myocardial infarction (MI) is a risk factor for recurrent coronary events and whether randomized treatment with pravastatin reduces that risk. Methods and Results —A nested case-control design was used to compare C-reactive protein (CRP) and serum amyloid A (SAA) levels in prerandomization blood samples from 391 participants in the Cholesterol and Recurrent Events (CARE) trial who subsequently developed recurrent nonfatal MI or a fatal coronary event (cases) and from an equal number of age- and sex-matched participants who remained free of these events during follow-up (control subjects). Overall, CRP and SAA were higher among cases than control subjects (for CRP P =0.05; for SAA P =0.006) such that those with levels in the highest quintile had a relative risk (RR) of recurrent events 75% higher than those with levels in the lowest quintile (for CRP RR=1.77, P =0.02; for SAA RR=1.74, P =0.02). The study group with the highest risk was that with consistent evidence of inflammation (elevation of both CRP and SAA) who were randomly assigned to placebo (RR=2.81, P =0.007); this risk estimate was greater than the product of the individual risks associated with inflammation or placebo assignment alone. In stratified analyses, the association between inflammation and risk was significant among those randomized to placebo (RR=2.11, P =0.048) but was attenuated and nonsignificant among those randomized to pravastatin (RR=1.29, P =0.5). Conclusions —Evidence of inflammation after MI is associated with increased risk of recurrent coronary events. Therapy with pravastatin may decrease this risk, an observation consistent with a nonlipid effect of this agent.

Inflammation, Pravastatin, and the Risk of Coronary Events After Myocardial Infarction in Patients With Average Cholesterol Levels

Objectives To determine the combination of drugs and vitamins, and their doses, for use in a single daily pill to achieve a large effect in preventing cardiovascular disease with minimal adverse effects. The strategy was to simultaneously reduce four cardiovascular risk factors (low density lipoprotein cholesterol, blood pressure, serum homocysteine, and platelet function) regardless of pretreatment levels. Design We quantified the efficacy and adverse effects of the proposed formulation from published meta-analyses of randomised trials and cohort studies and a meta-analysis of 15 trials of low dose (50-125 mg/day) aspirin. Outcome measures Proportional reduction in ischaemic heart disease (IHD) events and strokes; life years gained; and prevalence of adverse effects. Results The formulation which met our objectives was: a statin (for example, atorvastatin (daily dose 10 mg) or simvastatin (40 mg)); three blood pressure lowering drugs (for example, a thiazide, a β blocker, and an angiotensin converting enzyme inhibitor), each at half standard dose; folic acid (0.8 mg); and aspirin (75 mg). We estimate that the combination (which we call the Polypill) reduces IHD events by 88% (95% confidence interval 84% to 91%) and stroke by 80% (71% to 87%). One third of people taking this pill from age 55 would benefit, gaining on average about 11 years of life free from an IHD event or stroke. Summing the adverse effects of the components observed in randomised trials shows that the Polypill would cause symptoms in 8-15% of people (depending on the precise formulation). Conclusion The Polypill strategy could largely prevent heart attacks and stroke if taken by everyone aged 55 and older and everyone with existing cardiovascular disease. It would be acceptably safe and with widespread use would have a greater impact on the prevention of disease in the Western world than any other single intervention.

https://www.bmj.com/content/bmj/326/7404/1419.full.pdf

A strategy to reduce cardiovascular disease by more than 80

Aims/hypothesis
Increased intra-abdominal fat is associated with insulin resistance and an atherogenic lipoprotein profile. Circulating concentrations of adiponectin, an adipocyte-derived protein, are decreased with insulin resistance. We investigated the relationships between adiponectin and leptin, body fat distribution, insulin sensitivity and lipoproteins.

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Relationship of adiponectin to body fat distribution, insulin sensitivity and plasma lipoproteins: evidence for independent roles of age and sex.

Against a background of growing interest in more sensitive assays for quantifying various acute phase proteins, we evaluated the performance of recently developed tests for C-reactive protein (CRP), serum amyloid A (SAA) and mannose-binding protein (MBP) on the Behring nephelometer II (BNII). Sample results outside the calibration ranges of 3.5 to 220 mg/L for CRP, 3.3 to 215 mg/L for SAA and 0.09 to 5.6 mg/L for MBP were automatically re-measured at another dilution. The lower limits of detection were 0.01, 0.7 and 0.01 mg/L for CRP, SAA and MBP, respectively. The coefficients of variation (CV) for intra- (n > or = 20) and inter- (n > or = 15) assay precision were < 5.2% and < 8.5%, respectively, for the three proteins at concentrations representing low, normal and high. Linearity for each method was within 5% of the expected values throughout the calibration range. We observed no significant interference from bilirubin (up to 300 mg/L) or haemoglobin (up to 10 g/ L) for the three tests. Method comparison studies performed for CRP and SAA yielded the following results: y (CRP on BNII) = 0.75x (ELISA, Hemagen) -0.25 mg/L (r = 0.981, Sy/x = 2.1 mg/L; y (SAA on BNII) = 1.44x (ELISA, Hemagen) -9.9 mg/L (r = 0.972, Sy/x = 6.9 mg/L), where ELISA is enzyme-linked immunosorbent assay. Reference intervals established in 261 adult blood donors (aged 36.2 +/- 9.0 years) were found to be log-normal with 2.5th, 50th, and 97.5th centiles of < 0.17, 1.00 and 10.1 mg/L for CRP, < 0.84, 2.10 and 9.70 mg/L for SAA; and 0.30, 1.28 and 4.10 mg/L for MBP. We observed no relationship with CRP concentration and age; however, SAA levels increased with age while MBP levels decreased. The BNII provides a simple, rapid and sensitive system for measuring CRP, SAA and MBP in human serum.

https://journals.sagepub.com/doi/pdf/10.1177/000456329803500607

Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum.

Inflammation is associated with diverse clinical conditions accompanied by characteristic changes in serum levels of the acute-phase proteins that can be used to stage the inflammatory process and evaluate the impact of treatment. Some acute-phase proteins increase during inflammation, while others, such as albumin, transferrin, and transthyretin, decrease. The current study reports reference ranges for serum levels of albumin, transferrin, and transthyretin based on a cohort of over 124,000 Caucasian individuals from northern New England, tested in our laboratory between 1986 and 1998. Measurements were standardized against CRM 470 (RPPHS) and analyzed using a previously validated statistical approach. Individuals with laboratory evidence of inflammation (C-reactive protein of 10 mg/L or higher) were excluded. The levels of all three analytes varied by age, generally rising until the second or third decade of life and then decreasing thereafter. Albumin and transthyretin levels were higher during midlife among males as compared to females; the maximum being at 25 years for albumin (5%) and 35 years for transthyretin (16%). In contrast, above the age of 10 years, transferrin levels were increasingly higher among females (7% at 20 years). When values were expressed as multiples of the age- and gender-specific median levels, the resulting distributions fitted a log-Gaussian distribution. When patient data are normalized in this manner, the distribution parameters can be used to assign a corresponding centile to an individual's measurement simplifying interpretation. The ultimate interpretation of an individual's measurement relies upon the clinical setting.

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Reference distributions for the negative acute-phase serum proteins, albumin, transferrin and transthyretin: a practical, simple and clinically relevant approach in a large cohort.

Background: C-reactive protein (CRP) is a widely recognized indicator of inflammation and is known to play an important role in atherogenesis. Recent prospective studies have demonstrated that increased CRP concentrations within the reference interval are a strong predictor of myocardial infarction, stroke, sudden cardiac death, and peripheral vascular disease in apparently healthy adults. On the basis of available evidence, the American Heart Association and the CDC have issued guidelines for the utility of CRP in the primary prevention of coronary heart disease and in patients with stable coronary disease or acute coronary syndromes. Nevertheless, there remains considerable work to optimize the utility of this marker for risk assessment.

Issues: Most traditional CRP tests designed to monitor acute and chronic inflammation have inadequate sensitivity for risk stratification of coronary disease. Thus, manufacturers have had to develop tests with higher sensitivity. Because an individual’s CRP concentration will be interpreted according to fixed cut-points, issues related to the preanalytic and analytic components of CRP measurement must be considered and standardized where possible to avoid potential misclassification of cardiovascular risk.

Conclusions: Efforts to define performance criteria for high-sensitivity CRP applications coupled with growing awareness of the physiologic aspects of CRP most likely will lead to refinements in standardization, improved performance in quality-assessment schemes, and enhanced risk prediction.

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Preanalytic and Analytic Sources of Variations in C-reactive Protein Measurement: Implications for Cardiovascular Disease Risk Assessment

Apolipoproteins and ischaemic heart disease: implications for screening

To minimize differences in apolipoprotein measurements among laboratories and methods, a standardization program involving common suitable reference material is needed. The Committee on Apolipoproteins of the International Federation of Clinical Chemistry initiated a collaborative study for the standardization of test systems for measuring apolipoproteins (apo) A-I and B, with 25 company laboratories and three research laboratories involved in apolipoprotein analysis to: (a) evaluate calibration differences among the test systems; (b) evaluate whether comparability of the data can be achieved with the use of frozen serum pools to recalibrate the different systems; and (c) evaluate and select suitable candidate reference material. We used 26 test systems for apo A-I and 28 for apo B. Relatively modest differences were found in calibration for apo A-I, but very wide differences were observed for apo B methods. After uniform calibration, the overall among-laboratory CV for apo B decreased from 19% to 6%. Three lyophilized serum preparations for apo A-I and three liquid-stabilized serum preparations for apo B were selected for further evaluation as candidate international reference materials.

Thomas B. Ledue

Papers

Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum.

Reference distributions for the negative acute-phase serum proteins, albumin, transferrin and transthyretin: a practical, simple and clinically relevant approach in a large cohort.

Preanalytic and Analytic Sources of Variations in C-reactive Protein Measurement: Implications for Cardiovascular Disease Risk Assessment

Apolipoproteins and ischaemic heart disease: implications for screening

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B