Cell-Free DNA Biomarkers
Laboratory Rotation
Bios 703
Fall 2010
by Katherine Doyle
Advisor: Dr. Baranova
DNA Biomarkers have been the subject of research for decades. In this paper
we will discuss the cell-free DNA biomarkers, how they were discovered, and how these
discoveries have affected or could affect diagnostic medicine today. The paper will
briefly discuss the use of cell-free DNA biomarkers in viruses. Next we will explore the
many aspects of cell-free fetal DNA as well as the total cell-free DNA in pregnant
women. Finally we will briefly touch on several other conditions to which cell-free DNA
has been correlated.
History of Cell-Free DNA Biomarkers
The concept of being able to diagnose a disease with a simple blood test is often
the aim of researchers developing diagnostic tests for the medical field. Many types of
molecules have been screened in the blood of patients with various diseases to find
possible biomarkers that indicate not only the disease’s presence, but also the
progression of the disease. For example, many current diagnostic tests take advantage
of the antibodies the body develops to fight diseases and test for these antibodies
instead of for the disease itself.
Another concept which has been circulating the
scientific community for decades is the possibility of using circulating cell-free DNA as a
marker for diseases. Extracellular nucleic acids in circulation were first discovered in
1947 [1]. Several decades later, in the late 1970s researchers started quantifying the
free DNA in the serum of patients with cancer. These studies revealed that higher
levels of serum DNA was found in patients with cancer as compared with those that had
a benign disease. They also found that metastatic cancer patients had an even higher
amount of serum DNA [2-3]. However, these studies couldn’t determine if the DNA was
from the primary tumor because other diseases also demonstrated high levels of serum
DNA. A few years later scientist started to characterize the serum DNA of cancer
patients using strand stability assays.
Stroun and Anker demonstrated that this
circulating DNA was likely to be neoplastic [4].
Continuing research in the 1990s
allowed for the detection of mutated ras sequences in the serum of patients with solid
tumors [5] followed quickly by microsatellite analysis of the serum from patients with
small cell lung cancer and head and neck cancer. These studies showed that the
serum of patients with solid tumors contained circulating DNA with the entire range of
genetic changes found in primary tumors [6-7].
Carcinogenic Viruses
Another source of circulating DNA is foreign DNA from viruses and bacteria. The
life-cycle of many viruses lends itself to viral DNA being circulated in the host body. An
example of this was found in the 1990s with Epstein-Barr virus (EBV). EBV is a major
cause of nasopharyngeal cancer.
Integration of viral DNA is a critical step in the
transformation and proliferation of some of the early precursors to cancer. This fact led
researchers to reason that the viral DNA should also be found in the serum of patients
with primary tumors caused by the virus. Research confirmed that EBV DNA was found
in the serum of patients with nasopharyngeal cancer [8-9].
A more recent study tested the serum of uterine cervical cancer patients positive
for human papillomavirus 16 (HPV16) DNA for the presence of HPV16 DNA in serum
using quantitative real-time PCR. This study found that only 30% of the patients with
the disease possessed detectable levels of the DNA in their serum. Also, patients with
invasive cancer showed higher copy numbers of the HPV16 DNA than those with
cervical intraepithelial neoplasia or micro-invasive cancer. No HPV16 DNA was found
in the normal controls. These results suggest that the HPV16 DNA levels found in
human serum could be used as a biomarker of invasive cervical cancer, but would
probably not work on other types [10].
Biomarkers Related to Pregnancy
The presence of fetal DNA was first found in maternal plasma samples in 1997
[11]. This cell-free fetal DNA can be found circulating in maternal blood as early as 5
weeks during pregnancy and then quickly clears the maternal blood stream after the
birth. This fetal DNA only makes up 3-6% of the total cell-free DNA, but it is still the
subject of research to develop early non-invasive prenatal diagnostic tests especially for
genetic diseases [12].
Traditionally the placenta has been seen to form an
impermeable barrier between the pregnant woman and the fetus. However scientists
now know that intact fetal cells and cell-free nucleic acids circulate freely in the maternal
bloodstream [13]. Current testing includes non-invasive ultrasounds to monitor for fetal
anomalies and maternal blood tests and invasive tests like amniocentesis and chorionic
villus sampling. Ultrasounds are commonly used to screen patients for anomalies while
the invasive testing is commonly only used to diagnose suspected problems (or for
paternity testing). Cell-free fetal DNA was first discovered in maternal blood samples in
the 1990s and since this time research has focused on using this DNA to develop noninvasive tests. However, the amount of fetal DNA in a maternal blood sample is so
small that current technologies cannot extract pure fetal DNA. Because the cell-free
maternal DNA swamps the fetal DNA the techniques that can be used basically detect
the paternally inherited DNA of the fetus. This greatly limits the applications for cell-free
fetal DNA sequence analysis [14]. Much of the current research has therefore focused
on the quantity of both fetal and total cell-free DNA that is found in the blood.
Several studies have found significant correlations between the quantity of cellfree DNA found in maternal blood samples and the development of certain conditions
for the fetus and/or the mother. For example, it has been found that plasma from the
second trimester contains significantly higher amounts of cell-free fetal DNA if the fetus’
chromosome contains aneuploidies such as trisomy 21 [15]. On the other hand total
cell-free DNA in the mother’s blood during the second trimester was higher in patients
who developed preeclampsia later in the pregnancy [16].
There is also a marginal
correlation between serum PAPP-A and first trimester cell-free fetal and total DNA
levels found in the mother which may be helpful in early screenings for adverse
pregnancy outcomes [17].
The above examples are of papers which found that there are correlations with
the quantity of cell-free DNA, whether fetal or maternal, and specific conditions affecting
the fetus and/or the mother. However, there have also been a number of studies which
have found no correlation, at all. For example, the study involving PAPP-A correlating
with higher quantities of cell-free DNA also found no correlation between the amount of
DNA and several pregnancy related molecules [17]. Lapaire, et. al. found that there
was no correlation between cell-free DNA levels and maternal smoking [18], while in
another study they found that total cell-free DNA levels did correlate with maternal BMI
in the second trimester through delivery, but that BMI didn’t correlate with cell-free fetal
DNA levels [19]. Research into maternal cell-free DNA will no doubt continue and grow,
and as new technologies become available prenatal screenings will undoubtedly evolve
to include the information being discovered.
Other Conditions
So far we have focused on cancer and pregnancy research involving cell-free
DNA. This is because the largest amount of research has been focused in these areas.
However, cell-free DNA has been found to contain possible biomarkers for many other
conditions. This section will quickly touch on these studies.
Cell-Free DNA circulating in blood has been found to have limited usefulness as
a marker of infection in febrile patients. Normal concentrations of the cell-free DNA can
exclude and infection in these patients and if the quantity of this DNA is >10-fold over
normal the concentrations can be used to stratify the severity of an infection. The
extremely high concentrations show a high prognostic value predicting mortality.
Unfortunately, because these tests rely on the quantity of the DNA only, they only work
if there is no other reason for elevated cell-free DNA in the body. This found that the
usefulness of cell-free DNA as a marker for infection was at least as accurate as
procalcitonin and more efficient than C-reactive protein [20].
Neutrophil extracellular traps (NETs) are a novel innate immune response.
These NETs are composed of cell-free DNA derived from neutrophils combined with
neutrophil-derived proteins. These NETs contain proteolytic activity and can trap and
kill microbes. Once the NETs have performed their function their remnants float in the
plasma contributing to the overall levels of cell-free DNA detected in the blood. Logters,
et. al. explored the use of the NETs to diagnose septic arthritis. The NETs’ diagnostic
value was compared to white blood cells, synovial white blood cells, C-reactive protein,
j-IL-6, j-TNF alpha, j-IL-1 beta, and myeloperoxidase. NETs values from patients with
septic arthritis were significantly higher compared to patients with osteoarthritis or
noninfectious joint inflammation.
Once the NETs were given a cut-off point the
diagnostic value was evaluated and had a sensitivity of 0.89, a specificity of 1.0, a
positive predictive value of 1.0, and a negative predictive value of 0.97. These results
seem to suggest that the NETs hare a valuable marker for the diagnosis of septic
arthritis, although this will have to be confirmed with large clinical trials [21].
Due to the increase in cell-free DNA circulating in plasma during many types of
illness and injury this DNA it has been theorized that this DNA is associated with tissue
injury.
That makes cell-free DNA the perfect biomarker for exercise-induced
inflammation (athletic overtraining).
proportion to training loads.
In fact plasma DNA concentrations increase in
This suggests that cell-free DNA is a very sensitive
biomarker for the monitoring and quantification of overtraining in athletes [22]. If this
DNA is so sensitive to muscle damage due to excessive exercise it stands to reason
that it would be sensitive to muscle damage and death caused in other ways as well.
Since the quantity of circulating cell-free DNA has been associated with tissue
damage it is logical to assume that it is associated with cell damage. Thus researchers
decided to see if there was a correlation between cell damage due to hemodialysis and
cell-free plasma DNA.
They also wanted to see if the levels would correlate with
annexin V expression and 7-amino-actinomycin D nuclear staining of blood leukocytes.
They found that concentrations of DNA increased significantly after 20 min of
hemodialysis and that these concentrations stayed high after treatment stopped. Gel
electrophoresis showed ladders typical of apoptosis in the post-hemodialysis samples.
Overall the authors demonstrate that cell-free plasma DNA concentrations, annexin V,
expression, and 7-amino-actinomycin uptake in leukocytes increases.
Also, the
appearance of the increased plasma DNA in ladders typical of apoptosis and the 7amino-actinomycin in leukocytes suggests that most of the circulating DNA I these
patients originates from apoptotic leukocytes [23].
Another possible use for cell-free DNA is to detect the response of a patient to a
drug. Hydroxyurea is a drug used to treat acute pain in patients with sickle cell anemia
[24]. It has been shown previously that the acute painful episodes found in sickle cell
anemia patients are associated with a greater than 10-fold increase in cell-free DNA as
compared to steady state [25].
When these patients are treated for this pain with
hydroxyurea the quantity of cell-free DNA was significantly decreased. The authors
have suggested that these results may mean that hydroxyurea reduces the amount of
tissue damage and that this is what causes the decrease in pain. This discovery also
lends itself to the development of a monitoring system to optimize clinical effect of
hydroxyurea while minimizing the toxicity [24].
Conclusions
Overall, the quantification of cell-free circulating DNA has proved to have a
limited usefulness as a biomarker medium. Currently, the concentration of this DNA is
the most common type of analysis performed, however, the information on the makeup
of this DNA could prove to be more useful. This is especially true in pregnant women
where analysis of the fetal DNA would be extremely enlightening. Current technology
cannot isolate fetal DNA from the mother’s blood, but this will undoubtedly change in the
future, considering how useful this DNA already is in detecting possible complications in
pregnancies. The research into the possible uses for cell-free DNA as a diagnostic tool
are ongoing and will undoubtedly provide much more useful information to the
biomedical field in the future.
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