The Role of Nucleic Acids in Plasma, Serum
Nucleic acids circulating in plasma and serum can be screened for a variety of conditions. Testing fetal DNA found in maternal plasma may become a noninvasive diagnostic approach.
Published February 21, 2007
By Jill Pope
Academy Contributor
Most ninth-grade biology students can tell us that DNA and RNA are found within cells. But in both healthy and sick people, these nucleic acids can also be found circulating freely in plasma (the fluid in which blood cells are suspended) and serum. Scientists don’t yet understand exactly how and why nucleic acids are released into circulation, but these nucleic acids are proving to be useful as diagnostic tools in prenatal and cancer care.
Today, researchers are working toward noninvasive prenatal diagnosis of several disorders by analyzing fetal DNA in maternal blood. DNA markers can also aid in the diagnosis of cancer or tell doctors whether a person is responding to chemotherapy. Analysis of circulating RNA may also yield tumor markers and ways to detect fetal abnormalities and pregnancy complications.
No Conclusive Proof
At the same time, basic questions remain. Are DNA and RNA deliberately released into body fluids, or are they a byproduct of some other process? How do they enter the circulation? Peter Gahan of the University of London, along with Maurice Stroun and Philippe Anker, two of the field’s pioneers, have shown that there is a spontaneous release of both DNA and RNA from living cells, including tumor cells. This does not preclude other sources for nucleic acids in plasma and serum, however, such as apoptosis (programmed cell death).
“There are theories, and some evidence, but still no conclusive proof” as to the role circulating nucleic acids play in the body, says Ramasamyiyer Swaminathan, who served as editor, along with Peter Gahan and Asif Butt, of Annals of the New York Academy of Sciences Volume 1075, Circulating Nucleic Acids in Plasma and Serum IV. He organized and hosted the most recent conference on the subject, held at King’s College, University of London, in September 2005. More than 200 experts in the field attended, and this volume provides a record of the meeting.
Much of the research is geared toward developing better diagnostic tools. “I think that for things like lung cancer, where early detection is important, and conventional methods are unable to detect it, this will be very useful,” Swaminathan says. He also believes research will soon translate into maternal blood tests to diagnose prenatal disorders.
What Can Fetal DNA Tell Us?
Since the 1990s, scientists have been able to detect fetal DNA in the bloodstream of pregnant women. Circulating fetal DNA can be used diagnostically in two ways. Its quantity can be measured to aid in the detection of preeclampsia (pregnancy-related high blood pressure), risk of early delivery, and Down syndrome. Scientists can also examine the DNA qualitatively to look for the presence of certain genetic factors, such as those that indicate blood disorders such as β-thalassemia (severe anemia) or Rh disease.
Before doctors can tell if a pregnant woman has levels of fetal DNA in her bloodstream that are cause for concern, scientists need to establish a baseline for normal levels of fetal DNA in maternal blood. To do that, Diana Bianchi and her colleagues at Tufts-New England Medical Center investigated whether factors such as maternal age, weight, smoking, ethnic background, and type of conception affected circulating fetal DNA levels in normal pregnancies. They found that maternal weight in the second trimester was the only relevant factor—and that fetal DNA levels were lower in mothers who were heavier, which may have to do with the larger volume of body fluids in the heavier women.
Increased levels of fetal DNA in the mother’s bloodstream can be used to monitor pregnancy complications and may, in the not too distant future, help predict them. Bianchi’s group has found that among women at risk for delivering early, those with high concentrations of fetal DNA in their blood were significantly more likely to deliver before 30 weeks than those with lower levels.
The Role of Preeclampsia
Dennis Lo and colleagues at Prince of Wales Hospital in Hong Kong have found that preeclampsia is associated with a five-fold increase in fetal DNA levels. Both Lo’s group and Bianchi’s group have found that it is possible to detect trisomy 21, the chromosomal triplication that causes Down syndrome, by measuring levels of fetal nucleic acids in maternal plasma.
Adding to the progress in Down syndrome diagnostics, Vincenzo Cirigliano and colleagues at the General Lab in Barcelona, Spain reported that an alternative to karyotyping called quantitative fluorescent PCR could decrease the time needed to confirm the presence of an extra chromosome 21 in fetal DNA from two to three weeks to one or two days. Cirigliano’s group analyzed some 30,000 amniotic fluid samples, and found that the rapid technique was highly accurate in detecting major fetal abnormalities.
The ability to analyze fetal DNA within a maternal blood sample has already led to changes in clinical practice. Dennis Lo and his colleagues demonstrated in the late 1990s that a test of fetal DNA in maternal serum could reliably indicate whether the fetus has Rh-negative or Rh-positive blood. Mothers who are Rh negative need to find out their baby’s Rh status, because the baby may be at risk of developing Rh disease, in which the mother’s immune system attacks the baby’s blood cells. In parts of Europe, noninvasive maternal blood tests for fetal Rh status are now part of standard prenatal care.
Separation Anxiety
About 10 years ago, the discovery of fetal DNA in maternal plasma had many researchers excited about the potential to screen the DNA for genetic diseases and disorders without invasive procedures such as amniocentesis. Since that time, the problem has been how to distinguish fetal DNA from the maternal DNA around it. Until recently, the only reliable way to know the DNA belonged to the fetus was to detect a Y chromosome. Because females have two X chromosomes, if a Y chromosome were present, it would have to be from a male baby. (At-home baby gender tests that look for the Y chromosome are now on the market, but the tests are controversial.)
The picture is changing now, as researchers have reported two different ways to distinguish the baby’s DNA from the mother’s. One promising marker of circulating fetal DNA is its size. Sinuhe Hahn and colleagues at the University Women’s Hospital in Basel, Switzerland, have found that circulating fetal DNA molecules are measurably smaller than circulating maternal DNA molecules. Using gel electrophoresis, they observed that about 70% of cell-free fetal DNA was less than 300 base pairs in length, while about 75% of cell-free maternal DNA was more than 300 base pairs. They were able to separate out the fetal DNA by selecting for and enriching the smaller DNA molecules.
The Role of Methylation
Another technique to identify circulating fetal DNA takes advantage of the difference in the methylation state of maternal and fetal DNA. Methylation is an epigenetic factor, meaning that it influences the expression of genes without changing the actual DNA sequence. The process, which plays a major role in gene silencing, occurs when a cytosine base is modified by the addition of a methyl group. Sites called gene promoter regions can be undermethylated (hypomethylated), which may increase transcription levels, or overmethylated (hypermethylated), which may prevent gene transcription.
Lo’s group looked at the methylation state of placental cell DNA and compared it with the methylation state of DNA in maternal blood cells. They discovered that the maspin gene, a well-known tumor suppressor gene, is hypomethylated in the placenta and hypermethylated in the maternal blood cells. They then detected hypomethylated maspin sequences circulating in the plasma of pregnant women and observed that these sequences were rapidly cleared from the plasma after delivery, indicating that they were fetal DNA. Though the source of fetal DNA in maternal plasma has not been established, many researchers believe it comes from the placenta. Researchers expect that Maspin could be the first of many fetal epigenetic markers.
Improving Cancer Diagnosis
Analysis of circulating nucleic acids is also proving fruitful in cancer care. Investigators are analyzing nucleic acids to help detect cancers early, reduce the need for invasive biopsies, and identify people who are likely to respond to treatment.
Many researchers have focused on lung cancer, the leading cause of cancer death worldwide. Most lung cancers are not found until they are in advanced stages, in part because current measures—chest X rays and cytological sputum tests that look for abnormal cells under a microscope—are not useful for early detection. Research shows that analyzing circulating DNA for methylation of tumor suppressor genes and for genetic instability of microsatellites can improve the diagnosis of lung cancer.
Yi-Ching Wang of National Taiwan Normal University in Taipei and coworkers recently tested a panel of biomarkers for this purpose. They analyzed DNA markers in sputum samples from cancer patients and healthy individuals and compared them with those markers in tumor or normal lung tissue samples from the same people to see whether DNA from sputum pointed to the presence of cancer. Their work yielded seven useful diagnostic markers, including methylation of the tumor suppressor genes p16INK4a and RARβ. The authors suggest that testing for these markers could improve current diagnostic methods, and that markers of DNA methylation could become powerful diagnostic tools.
Predicting Response to Chemotherapy
Doctors who treat lung cancer have more chemotherapy options today than they did 10 years ago. They can try another option if they can determine early on that a drug or drug combination is ineffective, saving the patients precious time and sparing them from unnecessary side effects. The imaging techniques used to assess tumor mass are often not sensitive enough to detect changes until after several rounds of chemotherapy. Stefan Holdenrieder and colleagues at the University of Munich set out to discover whether blood markers could detect the tumor’s response much earlier.
To date, CYFRA 21-1, a serum protein marker, has been the strongest indicator of prognosis in non-small cell lung cancer. Holdenrieder and his group have shown that measuring levels of circulating nucleosomal DNA (the basic unit of packaged DNA, usually found in the nucleus of cells but also found in cell-free form) along with CYFRA 21-1 can identify patients who will respond to the first round of chemotherapy. In their most recent work, they asked whether the same two markers could be used to predict response even earlier—during the first round of treatment.
In a study of more than 300 people with advanced lung cancer, the researchers measured the levels of a number of biomarkers and of nucleosomal DNA to distinguish those patients whose tumors were in remission from those whose tumors were progressing. Higher concentrations of nucleosomal DNA and CYFRA 21-1 identified a subgroup of patients who were unlikely to respond to chemotherapy, and it identified them early—nucleosomal DNA was measured on the eighth day of therapy and CYFRA 21-1 was measured before the start of a second round of therapy.
Detecting Lung Cancer with Circulating Nucleic Acids
Out of a subgroup of 270 patients with good clinical status, 84 had cancers that progressed. The combination of markers correctly identified 30% of these patients as non-responders. If the markers had been used to manage treatment, they could have allowed a change of regimen for the non-responders before the start of the second round. Importantly, the markers did not point to any of the remaining 70% of the patients in this group whose tumors responded well to the initial treatment.
Indeed, research on using circulating nucleic acids to detect lung cancer may be ready to move to the clinic. A literature review in Clinical Chemistry (October 2006) found that based on what is now known, it would be possible to develop “a simple blood test” for screening, staging, prognosis, and evaluating response to treatment. The authors called for large studies “to integrate blood marker-based assays into the clinical setting.”
The next meeting devoted to circulating nucleic acids research will be held in May 2007, in Moscow. But before too long, Swaminathan predicts, this research will simply become part of the disciplines in which it is applied. Its techniques are already being adopted by specialists in fetal medicine, oncology, and other diseases. “I see that in a few years’ time, there will be a subsection of oncology conferences,” he says. “It is more important for oncologists to show other oncologists what is happening.” The research has already become a part of fetal medicine conferences. Wherever they share their findings, researchers in this field will continue to work toward earlier, faster, and more accurate diagnosis and management of disease.
Also read: The Primordial Lab for the Origin of Life
About the Author
Jill Pope writes about science and policy issues. She served as Senior Editor for The Cutting Edge: An Encyclopedia of Advanced Technologies (Oxford University Press, 2000).
