A Biopsy in a Tube–
The Tantalizing Promise of Circulating Cell-Free DNA

Kelly Grooms

Imagine if doctors could diagnose cancer, follow disease progression and monitor treatment results all through a simple blood test. No more subjecting patients to painful biopsies; no more using those biopsies to make treatment decisions years down the road. That is the tantalizing promise of liquid biopsies. Unlike traditional biopsies, which take a sample of tissue that—hopefully—includes cancer cells, liquid biopsies screen for free-floating pieces of DNA or RNA from the cancerous cells.

These extracellular DNAs and RNAs, called circulating cell-free nucleic acids (ccfNA), were first observed in human blood in 1948. Although these snippets of biological information come from a number of sources including normal cell death events, an elevated concentration of ccfNA can be associated with different diseases.  In the 1970s, researchers reported that the ccfDNA concentration in serum samples of individuals with breast cancer was elevated and that the concentration varied, depending upon disease progression and treatment (1) . These results were followed by research in the 1980s and 1990s that correlated the presence of ccfDNA in serum samples to malignancy of disease  (2) and then further identified mutated Ras molecules in serum samples taken from pancreatic cancer patients (3) (4) .

During development, tumor cells release their DNA into the blood circulation through apoptotic and necrotic cell death events as well as secretion from live cells. These events result in relatively high levels of ccfDNA in the blood of cancer patients. It also means that this population of circulating fragments found in a patient’s blood could act as biomarkers for their disease.

The Case for ccfDNAs as Biomarkers

Even heathy individuals have low levels of extracellular DNA circulating in their blood. However, most of the ccfDNA from healthy individuals belongs to Alu elements, the most abundant repetitive element in the human genome (5, 6). The gene sequences of ccfDNAs associated with disease are not the same as those found in the blood of health individuals. In addition, the amount of ccfDNA found is elevated during cancer development, and elevated ccfDNA levels in plasma have been associated with poorer survival rates for some types of cancer (7). All of these associations is what makes ccfDNAs such hopeful targets for liquid biopsies.

Challenges Associated with ccfDNA Biomarkers

Although there is a strong argument for ccfDNAs as blood-based cancer biomarkers for cancer, there are also some large obstacles. For one, the concentration of ccfDNA in blood is very low. This means any purification method needs to be able to isolate small fragments DNA from dilute samples. Because of the relatively low concentration of ccfDNA, genomic DNA contamination from lysed white blood cells can be a significant problem. As a result, how the blood is handled after it is collected plays an important role. For example, samples that are stored too long at room temperature or frozen and thawed before processing are more likely to have lysed white blood cells. In addition, the half-life of ccfDNAs in the blood is variable, but relatively short, ranging from 15 minutes to a few hours (7). This small window could make the timing for blood draws critical, especially when monitoring response to treatments.

Mutations have been detected in cancerous tissue and in the ccfDNA from corresponding plasma samples; however, none of these DNA alterations are absolutely cancer-specific, and in fact some of these sequences are also found in the ccfDNA of healthy individuals (8). This makes it challenging to interpret results correctly and raises the possibility of false positive results (9). These healthy and cancer-derived ccfDNA similarities also make it difficult to establish a clear baseline from which to judge future results.

Looking to the Future

The ideal biomarker would be close to 100% in both sensitivity and specificity to a disease state (8) . As the points highlighted above demonstrate, currently identified ccfDNAs cannot provide the needed level of sensitivity or specificity. However, as Kohler et al. points out, “The attractiveness of using ccfDNA as a biomarker lies in its non-invasive nature…” (8) Several studies have suggested that pairing ccfDNA with other well-known markers (e.g., prostrate-specific antigen (10); carcino embryotic antigen (11), or CA19-9 (12)) can improve overall sensitivity and specificity. In addition, future improvements in DNA isolation and detection technologies could improve the over all sensitivity of results with ccfDNA biomarkers.  

Cancer has been a scourge to medicine for centuries, and despite having the laser-like focus of scientist for the last several decades, it remains difficult to diagnose and treat. Part of the challenge is that cancer is not one disease, it is hundreds of diseases. These diseases vary greatly and as a result are diagnosed differently. There is not, and never will be, one test for “cancer”. However, noninvasive diagnostic tests using biomarkers like ccfDNA could go a long way towards making cancer easier to diagnose.


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