Diagnosing a solid organ disease using an accessible body liquid is a concept that dates back more than 3,500 years. Diabetes symptoms were noted in 1552 B.C., when Egyptian physicians documented frequent urination as a symptom of a mysterious disease that also caused emaciation. Ancient healers noted that ants seemed to be attracted to the urine of people with this disease. Centuries later, “water tasters” diagnosed diabetes by tasting the urine of people suspected to have the disease.
Whereas such rudimentary testing might be possible for diabetes, the diagnosis or monitoring of a complex remote cellular disease such as cancer poses a completely different challenge. However, the first observations of tumor cells in the blood occurred more than 100 years ago. In 1869, Thomas Ashworth examined the blood of a male patient under his microscope and found metastatic cancer cells, for which he noted, “One thing is certain, that, if they came from an existing cancer structure, they must have passed through the greater part of the circulatory system.”
Today, it is well established that various solid tumors can shed both intact cells or microvesicles, as well as cellular components, such as nucleic acids (resulting in cell-free DNA or RNA) in the bloodstream. Currently, three different sample sources for liquid biopsies are frequently used in clinical research and increasingly in clinical diagnostics.
Sample sources for liquid biopsies
For the above-mentioned circulating tumor cells (CTCs), various specialized methods for the enrichment of tumor cells or the depletion of non-tumor cells are commercially available. CTCs are usually either enumerated or analyzed with more advanced downstream molecular characterization technologies, such as reverse transcription polymerase chain reaction (RT-PCR) or sequencing. CTCs are rare, and only a few cells in several milliliters of plasma are typically found, even in metastatic cancer stages. Consequently, unbiased single-cell amplification technologies are sometimes useful before molecular characterization to increase the likelihood of detection.
As an alternative, liquid biopsies based on circulating cell-free nucleic acids have become popular in the last few years. Circulating cell-free tumor DNA is suspected to come mainly from dead tumor cells and has been shown in various studies to contain indicative cancer-related mutations.
A third and newer sample source, called the exosome, is the subject of growing interest, particularly in cancer research. Exosomes are microvesicles shed by organ cells as part of a biological communication system. 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.
All liquid biopsy approaches require a highly effective enrichment of the nucleic acids of interest. Together with a downstream analysis method that enables detection of the genomic similarities to the parent tumor and differentiation from normal tissue, the liquid biopsy approach is a very attractive option for cancer diagnostics. This is particularly true for diagnostic applications where traditional tissue biopsies have intrinsic shortcomings.
An alternative to biopsy
An estimated 1.6 million breast biopsies and one million prostate biopsies are performed each year in the United States.1 Tissue biopsies are invasive, whether performed during surgery, via a needle puncture, or as part of an endoscopic procedure. Additionally, these invasive approaches carry the risk of infection or other complications for the patient. Advanced lung cancer is a good example in this context, since a tissue biopsy often cannot be performed, even by a CT-guided fine needle technique, because the tumor or metastasis is sitting in an inaccessible part of the lung. Often, a tissue biopsy cannot be done because the patient’s poor condition makes the procedure too risky.
Even when a core needle biopsy or fine needle aspiration can be performed successfully, it is possible that after many pathological and histological examinations, there is not enough remaining tumor material to perform molecular companion diagnostic testing. Such tests guide treatment decisions for targeted therapies—for example EGFR and BRAF gene mutations or ALK fusions are relevant in various cancer types—and thus a lack of tissue or tissue of poor quality can limit treatment options. It is estimated that a tissue biopsy is not evaluable in up to 25 percent of cases of advanced non-small cell lung cancer (NSCLC), for example.
Recent large clinical studies now have clearly shown that, when a tissue biopsy is unavailable for any reason, blood can act as a surrogate for tissue samples to determine a predictive tumor biomarker and guide personalized treatment decisions. NSCLC is again a prime example, since a high specificity, sensitivity, and concordance to the tissue result was shown for the assessment of the EGFR mutation status from plasma samples before administration of certain tyrosine kinase inhibitors.2 Similar studies are ongoing for targeted therapies in various other cancers, such as colorectal and skin cancer.
In addition to predictive testing, liquid biopsy also can support disease prognosis. For example, CTCs have been detected in patients with several different epithelial cancers including breast, prostate, lung, and colon, and their clinical relevance has been demonstrated in studies correlating higher CTC counts with a negative cancer prognosis.
A role in disease monitoring
The area, however, in which liquid biopsies might have the highest impact in cancer care in the near future is disease monitoring. Liquid biopsies can monitor changes in tumor genetics over time, which is not possible with tissue biopsies. Where and when a resected tumor will reoccur cannot be predicted. Even with modern imaging technologies, it can be challenging to identify whether a lesion on a scan is a metastasis, and if it is, it cannot be re-biopsied frequently. The liquid biopsy approach might overcome those challenges and—in combination with the newest molecular characterization methods such as next-generation sequencing—holds great promise as a way to improve understanding of the fatal progression of the disease.
How realistic is such a scenario? In a recently reported study on lymphomas, a liquid biopsy-based test predicted recurrences more than three months before CT scan readings. Moreover, the liquid biopsy test was effective in identifying patients unlikely to respond to therapy.3 Another example is lung cancer; various targeted therapies against different mutation profiles are already available. Detailed molecular monitoring of the cancer over time could allow for detection of drug resistance earlier and thus for corrective action toward a personalized treatment regimen to be taken before the disease progresses. The liquid biopsy approach holds great promise in the ongoing effort to transform more cancers from lethal diseases to chronic conditions.
References
- Grady D. Study of breast biopsies finds surgery used too extensively. New York Times, February 18, 2011. www.nytimes.com/2011/02/19/health/19cancer.html. Accessed May 24, 2015.
- Douillard JY, Ostoros G, Cobo M, et al. Gefitinib treatment in EGFR mutated caucasian NSCLC: circulating-free tumor DNA as a surrogate for determination of EGFR status. J Thorac Oncol. 2014;9(9):1345–1353.
- Roschewski M, Dunleavy K, Pittaluga S, et al. Circulating tumour DNA and CT monitoring in patients with untreated diffuse large B-cell lymphoma: a correlative biomarker study Lancet Oncol. 2015;16(5):541-549.
