Liquid Biopsy – Dawn of a New Era in Diagnosis?

At a Glance

• Tissue biopsies are well-established in medical practice as a method for diagnosing cancer and other diseases, but they come with certain technical limitations
• Emerging technologies using blood or other body fluids could remedy those obstacles by unlocking molecular biomarker profiles of tumors, and improving diagnosis and treatment
• Liquid biopsy is one particular approach that is based on analysis of circulating free DNA (cfDNA), which has been shown to contain cancer-related mutations
• New research continues to emerge showing promise of this approach in the diagnosis of cancer and other diseases, as well as in ongoing treatment monitoring and management

Tissue biopsies are well established in medical practice as a means of diagnosing cancer and other diseases. Most patients go through the procedure as part of their solid cancer diagnosis; an estimated 1.6 million breast (1) and 1 million prostate biopsies (2) are performed each year in the United States alone. Biopsies are also frequently performed as part of a colonoscopy to diagnose inflammatory conditions like Crohn’s disease, before and after organ transplants, or when exploring potential causes of infertility.

While effective, this method of obtaining tissue samples does have certain limitations, predominantly because of their invasive nature – whether performed as a highly invasive surgical or as a fine needle biopsy. And since many biopsies tend to be burdensome and painful for patients, they can’t be used to regularly monitor the progress or recurrence of disease.

Getting enough tissue to perform all the necessary diagnostic procedures – including molecular biomarker testing to help guide treatment decisions for targeted therapies – can also prove difficult. This particularly applies to metastases of colorectal and skin cancer, or malignancies like lung cancers where, in approximately 25 percent of the cases, an evaluable biopsy sample cannot be obtained (3). And even when there is enough tissue, a traditional biopsy might not capture all of the relevant information because of the inherent heterogeneity of tumor tissue. Last but not least, tissue biopsies are associated with lengthy workflows; samples have to be fixed and the turnaround time for a report to the physician can take weeks. That’s too long for a cancer patient, especially for disease monitoring.

Taking the non-invasive route

Alternative technologies that use blood and other body fluids to diagnose cancers are, however, starting to emerge and appear to hold promise, not only in non-invasive disease diagnosis, but in treatment monitoring and more generally, in overcoming many of the shortcomings of the traditional approach. Liquid biopsy is one such technique (see infographic).

Broadly, liquid biopsy is a quick and comparatively straightforward process that usually includes a simple blood draw, extraction of nucleic acids from the blood plasma, and amplification of the molecular targets to enable analysis of the defined biomarkers. Reporting of results can be done within just a few hours.

Considering that liquid biopsies only recently started to make inroads into clinical practice, it is somewhat surprising that the principal concept of this technology is not new. In fact, the presence of cells in blood originating from a primary tumor was hypothesized more than 100 years ago (4). These circulating tumor cells not only indicate the presence of a tumor, but they’re also an early predictor of its spread to other parts of the body because of the role they play in metastases.

A more recent approach to liquid biopsy is based on analysis of circulating nucleic acids. Circulating free DNA (cfDNA) is believed to come mainly from dead cells and has been shown to contain cancer-related mutations (5). This phenomenon occurs in every person, but some physiological states and symptoms are associated with a significantly higher concentration of free-circulating nucleic acids; for example, after physical exercise, in pregnant women and in patients with cancers or autoimmune disorders. Research has shown that the quantity of circulating DNA in cancer patients is associated with tumor development – and this quantity decreases after surgery (6) – but it can also serve as an early prognostic marker for treatment failure (7).

One of the latest approaches to liquid biopsy focuses on nucleic acid detection from exosomes – small microvesicles that are used to transport genetic information within the body – sometimes described as the “Twitter of cells”. Each exosome can carry a tiny cargo of genetic instructions in the form of DNA and RNA molecules through the blood, urine or other fluids. The hope is that exosome detection could prove incredibly useful in diagnosing and monitoring diseases such as brain cancer, where the blood-brain barrier inhibits the emission of cfDNA into the blood stream.

Challenges in laboratory workflows

While biopsying using blood and other body fluids overcomes a host of obstacles associated with tissue biopsies, there remain some challenges. But, most of them can be effectively addressed by adjusting the laboratory workflow. I will explore the key considerations for cfDNA, which are the focal point of the work performed in our laboratory.

Large blood volumes

An important challenge when working with cfDNA is the low concentration of target DNA in plasma samples. So when performing liquid biopsies, larger volumes (10 to 20 ml) of blood must be drawn from the patient. While this might seem like a draw that is relatively high, it is important to remember the difficulty of obtaining tissue for a biopsy – particularly from those who suffer from advanced cancers. It is critical that the highest possible recovery of DNA is achieved to ensure the highest sensitivity in downstream analyses. And the higher the sensitivity, the higher the number of true positives captured.

Reducing the background

Proper handling and storage of samples is critical; background DNA can be released from white blood cells, for example, if the sample is not stabilized sufficiently. In addition to appropriate handling, special storage tubes can also help mitigate background DNA. Blocking oligonucleotides can help too by making sure that background DNA isn’t amplified or sequenced. Reduction of background ensures the appropriate sensitivity can be obtained.

Standardization and testing

To ensure appropriate DNA yield and purity, it’s important that a dedicated sample preparation is used on those samples found to have fragmented DNA. Yield and purity should also be maximized through process standardization and testing automation.

Clinical applications

Despite these challenges, liquid biopsies hold great promise, not only for patients, but for healthcare professionals too. Lung cancer is a prime example.

In 2014, the European Medicines Agency extended the label of AstraZeneca’s lung cancer drug Iressa to include patients tested with a liquid biopsy-based companion diagnostic. The corresponding CE-IVD marked commercial companion diagnostic launched shortly after by Qiagen.

In this instance, liquid biopsy technology is being used to detect EGFR mutations in patients for whom a suitable tissue sample cannot be obtained – this applies to as many as 25 percent of lung cancer patients (3). The presence of EGFR-activating mutations in a patient means he or she is more likely to benefit from Iressa treatment. It is anticipated that similar label updates for colorectal and skin cancer treatments will be made next and will be followed with new applications for liquid biopsy technologies. In fact the authors of a recent article in JAMA Oncology concluded that liquid biopsies could allow non-invasive EGFR targeting and monitoring on a repeated basis, in addition to the detection of other mutations (8).

As well as lung cancer, researchers are identifying biomarkers for glioma – one of the most common forms of brain tumor – that circulate in various body fluids (9), as well as glioblastoma (10). Their hope is to use those markers in much the same way that EGFR markers are used.

Another article published earlier this year (11) highlighted the potential of liquid biopsy to provide a new way to test for prostate cancer risk. The study identified men with prostate cancer with an accuracy of 83 percent and differentiated between men with prostate cancer and men with other non-malignant prostate disease with an accuracy of 90 percent.

In another recent study (12), researchers from Johns Hopkins University found that molecular analysis of a splice variant of the androgen receptor extracted from circulating tumor cells can tell if a patient is less likely to respond to two widely used drugs for metastatic prostate cancer.

Other research has demonstrated that changes to DNA circulating in the blood can be seen before related changes can be visualized using imaging technologies (13), suggesting that liquid biopsying might be used to help patients avoid harmful radiation.

The technique can also play a key role in monitoring the efficacy of treatment by detecting changes to the level of cfDNA. I also see an opportunity to monitor for genetic mutations associated with resistance to certain cancer treatments – cancer can evolve in response to a particular therapy, and monitoring these changes presents significant challenges, particularly because it now requires multiple invasive tissue biopsies. But researchers have shown that changes in the tumor genome can be identified through liquid biopsy technologies, which could complement current invasive tissue biopsies.

For instance, we have recently reported the results obtained during follow-up of two lung cancer patients during TKI treatment. While in one patient, who did respond to TKI treatment, the EGFR mutation was detected at similar levels in all plasma samples, in the other one the EGFR mutation disappeared from plasma DNA during treatment response, and reappeared at progression. These data suggest that the disappearance of circulating EGFR mutated DNA may be a marker of TKI response. This is being further evaluated at a large scale in patients with lung tumor, colorectal cancer and melanoma (14).

Currently, the highest utilization of liquid biopsy in clinical diagnostic practice today is, however, in non-invasive prenatal testing (NIPT) – the process screens for chromosomal and genetic abnormalities by taking advantage of the fact that fragments of fetal DNA circulate in the mother’s blood. This means that disorders like trisomy 21 and cystic fibrosis can be detected without an invasive amniocentesis, which can put the baby at risk.

A revolution in healthcare

Ongoing research is highlighting the great potential of liquid biopsy technology across a number of fields, and a growing number of combined initiatives of pharmaceutical and diagnostic companies to develop novel liquid biopsy-based companion diagnostics promises to expand the number of clinical applications in the near future.

While new applications are incredibly exciting, its use as a cancer screening tool is the most widely anticipated. It is estimated that around eight million people around the world die from cancer each year and research is showing the ability of liquid biopsy to detect it much earlier. This is hugely promising, in particular for highly lethal cancers, such as ovarian or pancreatic cancer, which today are almost always diagnosed too late. The ability to detect cancer early, perhaps even before symptoms appear, wouldn’t just revolutionize medicine, it could change the lives of millions.