Advances in Cell-Free DNA

Could one test really fit all?

Vol. 25 • Issue 4 • Page 22


A variety of patients are admitted to hospitals across the world every day-from expectant mothers going into labor to heart patients scheduled for bypass surgeries, even those making unexpected trips to the ED for chest pains or a broken arm. They’re dealing with different aches, varied diagnoses and are prescribed assorted medications. Solutions to all of their ailments may one day come by analyzing what’s unique to all of them.

Non-Invasive Prenatal Testing

Walking past the maternity floor, a new mother says she would take the place of her crying newborn and have her own heel pricked after delivery. Unfortunately, that’s not an option and heel pricks are essential to test a baby’s blood to screen for a set of medical conditions such as sickle cell disease and cystic fibrosis after delivery.

However, some pregnant women are getting their own blood taken to test their baby for an additional set of chromosomal conditions while still in utero by analyzing cell-free DNA (cfDNA). The process involves a blood sample containing both maternal and fetal DNA to be taken from the mother and checked for the presence of an abnormal amount of DNA from chromosomes 21, 18 and 13, which correlates to Down, Edward’s and Patau syndromes.1

This type of non-invasive prenatal testing (NIPT) is offered to high-risk pregnant women 35 and older or those who have had an ultrasound exam that shows a possible abnormality or have had another child with one of the chromosomal aneuploidies.

Chorionic Villus Sampling (CVS) is another test offered to high-risk pregnant women around 10-weeks gestation. Considered invasive, this test involves either inserting a needle through the abdomen and uterus and into the placenta or inserting a thin plastic tube through the vagina and cervix to obtain the placental DNA.2

Both CVS testing methods present little risk to the mother, but they do run the risk of causing a miscarriage-an estimated 1 in 100 women, according to NIH. This has, in turn, made NIPT more popular, especially when considering that more miscarriages occur in the over-35, high-risk group.

As NIPT technology develops, some physicians with a background in genetics are coming forward to remind pregnant women that cell-free DNA in prenatal testing does not have the ability to diagnose a condition. Rather, NIPT tells expectant mothers the likelihood their baby would be born with one of the birth defects being screened.

“There’s a prevailing misconception in minds of patients that NIPT can replace the use of diagnostic testing,” said Trilochan Saloo, MD, director of cytogenetics at CombiMatrix. “The misconceptions are unfortunately so high we don’t know whose responsibility it is to quell the misconceptions.”

A study conducted by CombiMatrix and presented by Saloo at the 2015 American Society of Human Genetics (ASHG) conference in Baltimore found a high number of false positives in NIPT. As a result, Saloo explained it’s important for women who get normal results to confirm them with a diagnostic test like amniocentesis (AFT).3

The non-profit Genetic Support Foundation is also dedicated to providing the public with an objective overview of NIPT.

“The technology is an amazing ­technology and a great advancement in medicine in general depending on how you look at it,” said Heidi Lindh, MS, CGC, executive director of Genetic Support Foundation. “But there are some limitations to it that we feel like are not always highlighted or made clear in all forms of the marketing or advertising.”

The invasiveness of follow-up testing could be seen as a hindrance for some mothers, but a false-positive result in NIPT could affect a mother’s decision to continue with the pregnancy or seek other options.

Cancer Detection and Treatment

Meanwhile, on the opposite side of the hospital, a 76-year-old man is getting a similar blood test done to test for cancer.

“The ability to use blood to answer the same sorts of questions can open up new opportunities, both in terms of potentially deciding which patients should get which therapies but also for the ability to monitor tumor development and look for resistance mechanisms and other mutations that might show up,” said Paul Spellman, PhD, a professor of molecular and medical genetics at Oregon Health and Science University (OSHU). “One very real possibility is you can monitor for the development of resistance mutations , and when you see them, you can switch therapies so I think this is something that will get rolled out clinically,” he said.

Traditionally, physicians or researchers would perform biopsies to test tumor tissue for cancer, but researchers have found that same information is released into the blood.

“Tissue biopsy is only single time and single site,” explained Chen-Hsiung Yeh, chief scientific officer of Circulogene Theranostics. “You are actually inserting a fine needle into the tumor mass trying to get a sample out and sometimes you will miss the tumor.”

And these “liquid biospies” can not only determine if a patient has cancer, but also be used to inform treatment. There’s still debate over which is the best liquid biopsy diagnostic, however-circulating tumor cells (CTCs) or cell-free DNA (cfDNA).

Researchers at OSHU conducted a study showing that whole-exome sequencing of cell-free DNA can find the same clinically relevant mutations identified in DNA from tumor tissue. This diagnostic option to inform cancer treatment has taken off because of next-generation sequencing.

However, a team of mechanical engineering researchers from Lehigh University, led by Yaling Liu, associate professor of mechanical engineering and mechanics, developed a new technique called “lab-on-a-chip” that isolates and detects CTCs at any form of the disease.

“Sequencing CTC after capture is trying to have a better molecular understanding of the nature of the CTC for diagnosis and treatment,” explained Liu. “It provides specific information about tumor/cancer cells.”

Companies like Roche and Circulogene have developed a way to give oncologists information about possible gene mutations.

“In a single drop of blood, we’re able to report back to the oncologists any mutations-approximately 3,000 mutations could exist across 50 known cancer-associated genes, and we’re able to report back to them if any of these mutations are detected,” explained Scott Rezek, chief operating officer at Circulogene. “We match that up to all known current FDA drugs that are on the market that target those genes, as well as any trials that are ongoing that target any of the mutations on those genes.”

One thing both sides can agree on is the fact that CTC and cfDNA liquid biopsy diagnostics have the potential to replace more-invasive biopsies as more studies are done. Their efforts are important steps in cancer research given that such a large portion of the population is dealing with a cancer diagnosis or is at risk. Nearly 1.6 million people were given a cancer diagnosis in 2015 alone, according to estimates from the NIH’s National Cancer Institute.

Providing Options

On the recovery room floor of the hospital is a 50-year-old woman who just underwent transplant surgery. She could also reap the benefits of circulating cell-free DNA.

Stanford University researchers determined a way to better determine whether a recipient rejects their transplant by analyzing a blood sample from the recipient, containing cell-free DNA of the donor. This method would eliminate the need for a more invasive cardiac biopsy, usually performed as a follow-up to the transplant surgery.7

Led by Stephen Quake, PhD, professor of bioengineering and applied science, the researchers studied 65 transplant patients and compared their biopsy and plasma results, which proved to be a more effective measure. As a result, molecular diagnostics company CareDx, for which Quake is a consultant, has licensed a patent from Stanford for one of the methods used in the study.8 They recently announced the completed expansion of their laboratory with next-generation sequencing (NGS) capability. Their new lab was built with the purpose of testing donor-derived cell-free.

Commercial, private and university laboratories continue to study cell-free DNA to provide less-invasive testing to patients. Whether a patient is getting checked for cancer, screened for a chromosomal abnormality in their unborn baby or is being monitored after a serious transplant surgery, the hope is that, with continued funding and research, patients will be able to understand the science of cfDNA and have it as an option for a hospital visit in the future.


  1.  ACOG. Cell-free DNA prenatal screening test. Available at:
  2.  NIH. Chorionic villus sampling. Available at:
  3.  CombiMatrix. New data from CombiMatrix study support follow-up diagnostic testing to confirm positive results from non-invasive prenatal testing. Available at:
  4.  Oregon Health & Science University. Knight Cancer Institute researchers track metastasis with cell-free DNA. Available at:
  5. PCLS. CertNDx Bladder Cancer Assay. Available at:
  6. ThermoFisher Scientific. Ion Personal Genome Machine (PGM) System. Available at:
  7. Stanford Medicine. New Stanford blood test identifies heart-transplant rejection earlier than biopsy can. Available at:
  8. CareDX. CareDx completes expansion of CLIA laboratory with next-generation sequencing platform capability. Available at:

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