Biomarkers for cardiovascular risk assessment

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LEARNING OBJECTIVES

Upon completion of this article, the
reader will be able to:

1. Identify inflammatory biomarkers related to cardiovascular disease.
2. Correlate Lp-PLA2 with atherosclerotic plaque.
3. Identify laboratory methods for measuring Lp-PLA2.
4. Describe genetic changes of cells resulting in tumors.
5. Identify biomarker factors useful to guide cancer treatment.

Research into
inflammatory biomarkers has opened up a new era in the assessment of
risk in patients with cardiovascular disease (CVD). Of the dozens of
candidate biomarkers, there are two that have accumulated sufficient
published evidence to support their utility in clinical practice:
high-sensitivity C-reactive protein (hs-CRP) and lipoprotein-associated
phospholipase A₂ (Lp-PLA₂).

Clinical review of Lp-PLA₂

Lp-PLA₂ is produced
predominantly by macrophages and is strongly associated with
rupture-prone plaque. Because it is produced by macrophages in
atherosclerotic lesions in the arterial intima, it is a more
vascular-specific marker than hs-CRP or other acute phase
reactant inflammatory markers, many of which are produced in the
liver.¹ Lp-PLA₂ is potentially linked to
the causal pathway of plaque inflammation, instability, and
eventual rupture; found at high levels in thin fibrous cap
atheroma; and can be lowered by lipid-modifying medications (statins,
fibrates, niacin, ezetimibe, and omega-3 fish oil).

Lipid-lowering therapies, including statins, are proven to reduce cardiovascular events, regardless of baseline LDL-C levels.

An elevated Lp-PLA₂ result may
indicate a need for more aggressive therapy, including treatment to
lower low-density lipoprotein cholesterol (LDL-C) goals. Lipid-lowering
therapies, including statins, are proven to reduce cardiovascular
events, regardless of baseline LDL-C levels. In multiple clinical
studies, Lp-PLA₂ has been shown to be a predictor of unstable
plaque, myocardial infarction (MI), and ischemic stroke.²
Since low-density lipoprotein has proven not to be a reliable
predictor of stroke, measuring levels of Lp-PLA₂ addresses
this unmet clinical need.

Lp-PLA₂ resides mainly on and travels
with LDL particles in plasma via apolipoprotein B binding, although it
is also associated with high-density lipoprotein, or HDL, particles,
lipoprotein (a), and remnant lipoproteins. Lp-PLA₂ is highly
upregulated in atherosclerotic plaque; and through hydrolysis of
oxidized LDL, this enzyme generates two pro-inflammatory mediators,
lysophosphatidylcholine and non-esterified oxidized fatty acid. In
pre-clinical animal studies, inhibition of the enzyme attenuates the
inflammatory process and slows atherosclerotic-disease progression. A
Phase II study sponsored by GlaxoSmithKline showed that a direct Lp-PLA₂
inhibitor (darapladib), in addition to standard-of-care treatment,
prevented expansion of the necrotic core, a region within coronary
plaque associated with a high risk of rupture.³

A substantial body of evidence supports Lp-PLA2 as a
cardiovascular risk marker that provides new information, over and above
traditional risk factors, to help identify individuals at increased risk
of suffering a heart attack or stroke.

The Lp-PLA₂ difference

Numerous peer-reviewed publications have
confirmed that elevated plasma levels of Lp-PLA₂ are
independently associated with risk of coronary heart disease
(CHD) and ischemic stroke. The Atherosclerosis Risk in
Communities, or ARIC, study showed that in individuals with
normal LDL, elevated Lp-PLA₂ levels were strongly
associated with heart disease and ischemic stroke, independent
of traditional risk factors and hs-CRP.^4,5
Elevated levels of both inflammatory markers conferred an even
higher risk of MI and stroke. Individuals with elevated Lp-PLA₂
and hs-CRP levels had greater than a fourfold increase in risk
for heart attacks, and more than an elevenfold increase in risk
for ischemic stroke. Additionally, increased levels of Lp-PLA₂
doubled the risk of ischemic stroke at every level of systolic
blood pressure, while individuals with the highest levels of
Lp-PLA₂ and elevated blood pressure had nearly a
sevenfold increase in risk of suffering an ischemic stroke.⁶
In the KAROLA study, high-risk patients followed for four to six
years showed a significantly lower incidence of cardiovascular
events if their Lp-PLA₂ levels were 7

Lab measurement of Lp-PLA₂

Testing for Lp-PLA₂ in the laboratory
is available in an ELISA test format or as an automated format. Two
highly specific monoclonal antibodies are used in the assay, and it is
calibrated to a well-characterized recombinant Lp-PLA₂
standard to increase the accuracy of the test. The automated assay
employs immunoturbidimetric technology and can be run on the Hitachi,
Roche Modular P and Olympus analyzers. Additional applications are in
development. The Lp-PLA₂ protein in serum is generally stable
(i.e., the protein itself does not degrade), but it is highly
recommended that the serum and plasma samples be collected and stored
according to the Recommended Specimen Collection and Storage procedures.

Acknowledging the limitations of traditional risk
factors to precisely assess cardiovascular risk across the general
population, the National Cholesterol Education Program Adult Treatment
Panel, or NCEP ATP III, report recognized the potential of inflammatory
markers to help refine cardiovascular risk assessment. As Lp-PLA₂
evaluates vascular inflammation specifically, persons with
elevated levels of Lp-PLA₂ could potentially be classified
into a higher risk category, prompting the need to further intensify
lifestyle and medication therapy in direct proportion to the degree of
determined risk.^8-11

An elevated Lp-PLA2 result may indicate a need for more
aggressive therapy, including treatment to lower low-density lipoprotein
cholesterol (LDL-C) goals.

The current literature has reported that the
central 90th percentile of Lp-PLA₂ levels range from 120 to
342 ng/mL for women and 131 to 376 ng/mL for men.¹² Recently,
using data from all currently published Lp-PLA₂ studies, an
independent consensus panel of cardiologists, neurologists and
laboratorians endorsed a cut point of >200 ng/mL to identify patients at
higher risk for CHD/CVD.¹³

The same consensus panel recommended, consistent
with the ATP III guidelines, that Lp-PLA₂ should be used as
an adjunct to traditional risk-factor assessment. They suggested that
elevated Lp-PLA₂ levels would justify more aggressive
risk-reducing strategies, including treatment to lower LDL-C goals.

In summary, a substantial body of evidence
supports Lp-PLA₂ as a cardiovascular risk marker that
provides new information, over and above traditional risk factors, to
help identify individuals at increased risk of suffering a heart attack
or stroke. The level of the enzyme in the bloodstream is related to the
progression of instability of the atherosclerotic plaque, and the
likelihood for plaque rupture and a resulting thrombotic event. As such,
Lp-PLA₂
should be used as an adjunct in persons assessed to be at moderate or
high cardiovascular risk by traditional risk factor assessment, to help
refine absolute risk status and identify the individuals who would most
benefit from intensification of lifestyle modification and lipid
lowering therapies.

Robert L. Wolfert, PhD, is executive vice
president and chief scientific officer at diaDexus Inc. in South San
Francisco. For more information, please visit
www.diadexus.com
.

Note: This article is followed by another
article, “Cancer biomarkers — a good start,” that is also part of the
Continuing Education test.

References

1. McConnell JP, Hoefner DA. Lipoprotein-associated phospholipase A₂.
   J Clin Lab Med. 2006;26:679-697.
2. Garza CA, et al. Association between lipoprotein-associated
   phospholipase A₂ and cardiovascular disease: a systemic
   review. Mayo Clin Proc. 2007;82(2):159-165.
3. Serruys PW, et al. Darapladib: effects of the direct
   lipoprotein-associated phospholipase A₂ inhibitor
   darapladib on human coronary atherosclerotic plaque. Circulation.
   2008;118:1172-1182.
4. Ballantyne CM, Hoogeveen RC, Band H, Coresh J. Folsom AR, Heiss
   G, Sharrett AR. Lipoprotein-associated phospholipase A₂,
   high-sensitivity C-reactive protein, and risk for incident heart
   disease in middle-aged men and women in the Atherosclerosis Risk in
   Communities (ARIC) study. Circulation. 2004;109:837-842.
5. Ballantyne CM, 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;165:2479-2484.
6. Gorelick PB. Lipoprotein-associated phospholipase A₂
   and risk of stroke. Am. J Cardiol. 2008: 101[suppl]:34F-40F.
7. Koenig W, Twardella D, Brenner H, Rothenbacher D.
   Lipoprotein-associated phospholipase A₂, predicts future
   cardiovascular events in patients with coronary heart disease
   independently of traditional risk factors, markers of inflammation,
   renal function and hemodynamic stress (KAROLA). Arterioscler
   Thomb Vasc Biol. 2006;26:1586-1593.
8. Expert Panel on Detection, Evaluation, and Treatment of High
   Blood Cholesterol in Adults. Executive summary of the 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). JAMA. 2001;285:2486-2497.
9. National 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;106:3143-3421 II-30-II-31.
10. Pearson TA, Mensah GA, Alexander RW, Anderson JL, 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;107:499-511.
11. Smith SC, Allen J,, Blair SN, Bonow RO, Brass LM, Fonarow GC, et
    al. AHA/ACC guidelines for secondary prevention for patients with
    coronary and other atherosclerotic vascular disease: 2006 update.
    Circulation. 2006;113:2363-2372.
12. Brilakis ES, McConnell JP, Lennon RJ, Elesber AA, Meyer JG,
    Berger 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.
    Eur Heart J. 2005;26:137-144.
13. Davidson MH, Corson MA, Alberts MJ, et al. Consensus Panel
    Recommendation for Incorporating Lipoprotein-Associated
    Phospholipase A₂
    Testing Into Cardiovascular Disease Risk Assessment Guidelines.
    Am. J Cardiol. 2008: 101 [suppl]:51F-57F.

Cancer biomarkers — a
good start

By Stephen Little, PhD

Cancer researchers
and the pharmaceutical industry have invested trillions of dollars and
millions of man years in the search for drugs that will cure cancer; but
despite this gargantuan effort, success to date has been patchy. In
2009, there is a sense of “could do better” rather than the conclusive
triumph over cancer that everyone would hope for. Why is this, and how
can the situation be improved?

Perhaps part of the problem is the remarkable
power of cancer cells to evolve in response to their environment. The
pharmaceutical industry is incredibly good at developing new drugs with
the ability to kill cancer cells; but, often, when these are
administered to a patient, there is an initially positive response until
the cancer mutates and evolves in an effort to establish a way to
overcome the effects of the therapy.

The plasticity of the cancer genome means that it
is not sufficient to kill a cancer as it exists at one point in time —
there is a need for a dynamic approach that follows the cancer as it
twists and turns to escape the effect of the therapy, and to hunt it
down until every last cell is destroyed. This, of course, is easier said
than done.

There are, however, grounds for optimism. The
Human Genome Project is complete, the Cancer Genome Project is underway,
and our understanding of how cancer works has never been better. At a
basic level there are three types of genetic change responsible for a
normal cell turning into a tumor:

• activation of oncogenes — genes which tell the new cancer to
  grow;
• loss of tumor suppressors — genes which would have told the
  cancer to stop growing; and
• loss of DNA repair genes — genes normally work to maintain the
  integrity of the genome; without them, the other changes are much
  more likely to occur.

A common analogy is to liken the cell to an
automobile. The accelerator pedal represents the oncogenes, which, when
activated, is like locking the pedal in the “fully on” position. The
brake pedal is likened to the tumor suppressors; the loss of these means
that the car cannot stop. Perhaps it is stretching the analogy a little,
but an incompetent car mechanic would represent the loss of DNA repair
genes like that having an inept individual working on the car would
cause an increase in the likelihood of brake or accelerator problems.

It follows then, that to truly wipe out cancer
cells within the body, it is not enough to have effective drugs that
target some of the cancer-growth pathways — it is also essential to have
a way of monitoring the cancer itself, so the drug therapy can be
adjusted to match the tumor as it evolves. In this way, it might be
possible to use a sequence of treatments to allow a better outcome for
the patient.

The tools to allow this are now emerging in the
form of cancer biomarkers. The word “biomarker” has a very broad
definition and is essentially anything associated with drug response
that can be measured. This includes predictive biomarkers — which can be
used to select patients — and response biomarkers which (as their name
suggests) indicates whether a drug is working or not. There are many
classes of biological molecules which can be tested as biomarkers. The
most fundamental for cancer are genetic biomarkers, because cancer is
essentially a genetic disease in that it is caused by somatic gene
changes. Somatic gene changes are the underlying alterations which can
predict how an individual tumor will respond to treatment and include
mutations, methylation changes, gene rearrangements, and gene-expression
changes. All of these genetic changes cause a plethora of other
variations to the cell, its immediate environment, and to the whole
body; so, as a result, other biomarkers include proteins, peptides,
carbohydrates, and metabolites.

It follows then, that to truly wipe out cancer cells within the
body, it is not enough to have effective drugs that target some of the
cancer-growth pathways…

For a biomarker to be successful in guiding
future drug treatments, there are two key requirements. The first is
obviously that the marker, whatever it is, must be associated with drug
response. In the language of diagnostics, it must show clinical utility;
or, in common parlance, it must answer the “so what” question. If there
is no clearly defined treatment decision based on the use of the
biomarker, then it was probably not worth testing for in the first
place.

The second requirement has much more to do with
the practicality of implementing a biomarker-driven drug selection
strategy. Normally at the start of treatment, there will be a tumor
biopsy available — this is excellent material for the measurement of
many types of biomarkers and is probably the ideal sample. There is,
however, a major problem with the tumor biopsy. Earlier in this article,
it was pointed out that it would be essential to have a way of
monitoring the tumor as it evolved. Unfortunately, for most cancers,
there is no practical and safe way to take repeated primary biopsies, so
it becomes essential to be able to use a different source of tumor
material or even a surrogate for the tumor itself.

There is currently a great deal of interest in
the detection of circulating tumor cells¹ or circulating
nucleic acid which has been shed from the cancer. These methods can be
technically demanding and suffer from a lack of sensitivity, but they do
have the huge advantage that regular sampling is both feasible and
practical. If these methods can be honed to a workable level, the door
can be opened to biomarker monitoring and therapy adjustment.

There is also the possibility of using a
surrogate biomarker. For example, hair follicles have the same
epithelial origins as many cancers and can be used to indicate whether
or not a drug is having the expected effect within the body.

The use of biomarkers to guide therapy is still
in its infancy; and although there have been a number of notable recent
successes such as the use of KRAS mutation status² to guide
the use of the colorectal-cancer drugs Erbitux (cetuximab)^3,4,5,6
and Vectibix (panitumumab),⁷ and the association between EGFR
mutations and response to Iressa (gefitinib)^8,9,10 and
Tarceva (erlontinib)¹¹, there is still a long way to go
before biomarkers become part of the complete cancer-treatment regime. A
greater use of cancer biomarkers is not the only innovation needed to
improve outcomes with cancer drugs; but, perhaps, with their increasing
adoption, we will soon be able to report that cancer treatment has
improved from “could do better” to “a good start.”

Stephen Little, PhD, is the CEO of DxS Limited (www.dxsdiagnostics.com),
Manchester, UK.

Note per the author:  Erbitux
– trademark of Merck KGaA/Imclone Systems; Vectibix – trademark of Amgen
Inc.; Iressa – trademark of AstraZeneca group of companies; and Tarceva
-trademark of OSI Pharmaceuticals.

References

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   Epidermal Growth Factor Receptor Mutation in Transbronchial Needle
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2. Jimeno A, Messersmith WA, Hirsch FR, Franklin WA,Eckhardt SG.
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4. Van Cutsem E, et al. K-RAS status and efficacy in the first-line
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   without cetuximab in first-line advanced colorectal cancer, the
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6. Tejpar S, et al., Relationship of efficacy with K-RAS status
   (wild type versus mutant) in patients with irinotecan-refractory
   metastatic colorectal cancer (mCRC), treated with irinotecan (q2w)
   and escalating doses of cetuximab (q1w): The EVEREST experience
   (preliminary data). J Clin Oncol. 26: 2008. (May 20 suppl;
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7. Amado RG, et al. Analysis of K-RAS mutations in patients with
   metastatic colorectal cancer receiving panitumumab monotherapy.
   Paper presented at: European Cancer Organization (ECCO), May 24-26,
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8. Kimura H, Kasahara K, Kawaishi M, et al. Detection of Epidermal
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   Response to Gefitinib in Patients with Non Small-Cell Lung Cancer.
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