The 'Liquid' Biopsy

The advent of "precision" medicine is characterized by many new improvements in diagnostic, prognostic, and therapeutic methods and regimens. As part of this initiative, there has been a focus on minimizing the amount of tissue needed for testing via less invasive and safer procedures. One method that has drawn considerable interest in recent years is the "liquid biopsy". While definitions vary on the precise meaning of this term, it can broadly be thought of as collection of a body fluid sample to test for relevant biomarkers to inform patient management. It is most commonly applied to the collection of peripheral blood for analysis of cell-free circulating tumor deoxyribonucleic acids (DNA).

The first description of circulating DNA free from cells in human blood was in 1948, but this garnered little attention in the broader scientific community ². In 1977, scientists identified the presence of abnormally high levels of cell-free DNA (cfDNA) in the plasma and serum of cancer patients relative to health control patients and this cfDNA was presumed to represent mainly circulating tumor DNA (ctDNA)^1,5. Since this original description, other research has found that increased cfDNA generally reflects a multitude of pathologic processes, including malignant and benign neoplastic conditions, inflammatory diseases, stroke, trauma, and sepsis[2][11]. During these processes, nucleic acids may be shed into the blood by apoptotic and necrotic cells or by controlled secretion by living cells^2,11. While cell-free DNA is often used synonymously with circulating tumor DNA, one should remember that circulating non-tumor and non-human DNA may also be present in a sample.

While the current focus is dominated by DNA, additional components of a liquid biopsy include ribonucleic acids (RNA), circulating tumor cells (CTCs), extracellular vesicles (EVs), and tumor educated platelets (TEPs). The latter components are mainly of research interest. As more translational research is performed, these additional components of a liquid biopsy may have increasing clinical use in the future. A review of these elements is beyond the scope of this article and the reader is referred to the article by Batth et al for further reading.

Technological Considerations

Until recently, the available technology has not been sensitive enough to detect ctDNA and put it to meaningful use. Unlike molecular assays performed on body fluids for the detection of nucleic acids from viruses and other microorganisms which benefit from relatively large quantities of nucleic acids, circulating tumor nucleic acid fragments present at a fraction of the normal non-tumor cfDNA. ctDNA are usually small DNA fragments in the range of 140-170 base pairs (bp)³, and tumor type, progression, burden, proliferation rates, and therapy all affect the amount of ctDNA in a sample. In addition, although ctDNA is relatively stable in plasma and serum, it is removed from circulation by the liver and kidneys within hours ^3,4. Nevertheless, advances in pre-analytic processes and purification methods have allowed successful capture, amplification, and sequencing of ctDNA.

Methods that are currently used to detect or measure ctDNA can broadly be divided into two categories: polymerase chain reaction (PCR) based and next-generation sequencing (NGS) based. PCR-based assays generally have a faster turnaround time and are less expensive, but typically can only assess one or a few specific mutations at a time (limited multiplexing capability). PCR-based approaches can be further subdivided into methods that enrich for mutant sequences relative to wild-type (non-mutant) and those that achieve quantitation by compartmentalization. An example of the latter group is "digital PCR" which is becoming widely used for detection and quantification of specific, known mutations in ctDNA. The PCR is compartmentalized into thousands of tiny individual reaction volumes, either on a chip or by creating water-in-oil droplets. Each compartment or droplet either contains a targeted template fragment or not and produces a fluorescent signal if an appropriate template fragment is present within that volume. By counting the individual fluorescent volumes, it is possible to estimate the number of specifically targeted template molecules in the sample. For multiple targets tested at once (multiplexing), different fluorescent signals can be assigned to specific variant sequences.

Next-generation sequencing-based approaches permit assessment of a much broader range of possible mutations because sequencing can detect mutations occurring anywhere within the captured regions. To target mutation-prone regions of the genome, NGS libraries can be prepared from plasma DNA using either ligation/hybrid capture methods or targeted PCR enrichment methods. Variant allele fractions are generally much lower in liquid biopsies than tissue biopsies, often <1%, so regions of interest must be sequenced more deeply than in NGS of primary tumor tissue. In addition, extensive optimization of pre-analytic and analytic processes need to be done to maximize input sample and reduce PCR and sequencing errors. NGS approaches have the important advantage of being able to achieve much broader mutation coverage (simultaneous analysis of thousands of possible mutations). Thus, prior knowledge of the tumor's specific mutations is not needed. However, compared to simpler PCR-based methods, NGS techniques are more expensive, time consuming, and more technically challenging.

Advantages and Disadvantages

The advantages of liquid biopsies lie predominantly in the much less invasive nature of the procedures to obtain them relative to standard tumor biopsies. Consider, for example, the process involved to biopsy a lung mass. If the mass lies in a location that is accessible by interventional radiology or surgical biopsy, there is a risk of pneumothorax or hemorrhage, notwithstanding the cost of maintaining an operative suite within which to perform the operation. Liquid biopsies also allow more frequent and serial samplings over time to provide greater resolution to the behavior of tumors as well as their response to therapy. For example, in one study colorectal cancer patients who later radiographically demonstrated a good response to treatment had a drop of >90% of the ctDNA levels after the first 2 weeks of treatment ⁹. This has been shown to stratify risk of recurrence after resection with curative intent. In another study, breast cancer patients with detectable ctDNA after resection had a 25-fold higher hazard risk of recurrence ¹⁰. These concepts are analogous to testing that is currently done for hematologic malignancies, such as chronic myelogenous leukemia (CML) and serial testing for the presence BCR-ABL fusion transcripts. Finally, in instances when a tissue biopsy is not available, molecular profiling of tumors may still be performed via liquid biopsy.

Important disadvantages of liquid biopsy include the need for an initial histologic diagnosis to be obtained by tissue biopsy. Laboratories performing these assays need to be mindful of appropriate test utilization and the potential for "over interpretation" in the clinical context. The low variant frequency within the peripheral blood may lead to higher false negative rates and require significantly greater technical efforts and expertise to obtain reliable results.

Current and Emerging Applications

The clinical use of the liquid biopsy has significantly increased since 2014, when the first commercially available multigene liquid biopsy platform became available. Several assays are commercially available and FDA-approved, and some are considered as sufficient for treatment eligibility by insurance companies. For example, in 2016 the FDA approved the cobas® EGFR Mutation Test v2 to determine the eligibility of non-small cell lung cancer patients to receive certain EGFR tyrosine kinase inhibitors [7]. Adoption of the liquid biopsy to exclude patients from targeted therapy has seen much slower clinical adoption, mostly due to concerns for false negatives and generally accessible tumor tissue ³. Increasing clinical utilization has also been driven by patients and physicians wanting to identify targetable mutations for off-label use or clinical trial enrollment.

Emerging uses for liquid biopsies include their use as a complement to tissue biopsy mutational profiling, assessing treatment response, residual disease monitoring, detection of disease recurrence, and monitoring for emergence of resistance mutations ³.

Future Directions

The expected cancer-specificity of mutations makes ctDNA an attractive biomarker for early detection of cancer, which could have tremendous impact on patient care. However, since early-stage tumors are known to release very little DNA, there are many technical challenges to be overcome. Although liquid biopsy may be an attractive tool for cancer screening in asymptomatic patients, such applications will need to be carefully and thoughtfully considered to avoid excessive patient suffering and cost due to false positive results. In the short term, liquid biopsies may be more helpful to confirm malignancy in patients with already clinically or radiographically apparent lesions.

Conclusion

The literature focusing on ctDNA testing is quickly expanding and evolving. Ongoing areas of investigation include pre-analytic processes, factors affecting ctDNA detection rate, and prospective clinical trials. We are likely to see a more prevalent role of ctDNA in clinical care, as continued research on CTCs, cfDNA/RNA, and extracellular vesicles will provide increased resolution to the snapshot of tumor status obtained through liquid biopsies ⁴.

References

1. Chakravadhanula M, Tembe W, Legendre C, et al. Detection of an Atypical Teratoid Rhabdoid Brain Tumor Gene Deletion in Circulating Blood Using Next-Generation Sequencing. J Child Neurol 2013;29(9):NP81-NP85.
2. Schwarzenbach H, Hoon DSB, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011; 11: 426-37.
3. Batth IS, Mitra A, Manier S, et al. Circulating tumor markers: harmonizing the yin and yang of CTCs and ctDNA for precision medicine. Ann Oncol 2017;28(3):468-477.
4. Best MG, Sol N, Zijl S, et al. Liquid biopsies in patients with diffuse glioma. Acta Neuropathol 2015; 129: 849–865.
5. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the Serum of Cancer Patients and the Effect of Therapy. Cancer Res 1977 March; 37: 646-650.
6. Patel AA, Chandra P. Emerging Concepts on Liquid Biopsy: Circulating Tumor in Cell Free DNA (cfDNA). Short Presentations on Emerging Concepts. College of American Pathologists, 6 Sept 2016. World Wide Web. Accessed 12 August 2017.
7. "cobas^® EGFR Mutation Test v2 - P150047". Medical Devices. U.S. Food and Drug Administration, 9 Sept 2016. World Wide Web. Accessed 20 Sept 2017.
8. Bettegowda C, Sausen M, Leary RJ, et al. Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Sci Transl Med 2014 Feb 19;6(224):224ra24/
9. Tie J, Kinde I, Wang Y, et al. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann Oncol 2015; 26: 1715-1722.
10. Schiavon G, Hrebien S, Garcia-Murillas I, et al. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer. Sci Transl Med 2014 Nov 11;7(313):313ra182.
11. Diaz LA Jr, Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. J Clin Oncol 2014 Feb 20; 32(6):579-86.

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