Liquid biopsy: a perspective for probing blood for cancer

Cancer is a highly complex set of diseases that develop common but evolving pathophysiologic characteristics and processes. With appropriate intrinsic and extrinsic biological conditions, including the influence of supportive microenvironmental, biomechanical, and immunologic conditions, tumor cells and/or associated endothelial cells, tumor cell products such as nucleic acids, and extracellular vesicles may shed and potentially intravasate into blood and/or lymphatic vessels, ultimately leading to seeding and reseeding of metastatic foci. Molecular phenotypes of metastases may be impacted by intratumoral heterogeneity of primary and metastatic tumors and evolutionary processes that influence physiology and metabolism of metastatic tumor cells and their drug sensitivity. Moreover, during drug treatment of heterogeneous tumors, sensitive cancer cells may be ablated, while residual cancer cells that may be resistant to a given therapy survive and multiply, thereby contributing to further evolution of metastases into diverse tumor cell phenotypes. To combat this, cancer treatment has become increasingly tailored and evermore dependent on the identification of biomarkers that predict therapeutic response.

The need to serially sample the changing and evolving landscape of cancer in real-time throughout the course of disease is being addressed by the field of liquid biopsy, whereby tumor cells or tumor cell products present in blood or other bodily fluids that can be easily accessed with minimal morbidity are identified, measured, and characterized. The field has presented major opportunities for engineers and bioengineers, cancer biologists, cancer clinicians, genomic scientists, informaticians, and other scientists to converge into effective multidisciplinary teams for the development of new technologies that will ultimately impact the care of cancer patients.

Although the term liquid biopsy was initially used primarily to describe the identification of circulating tumor cells (CTCs) in blood samples, liquid biopsy now encompasses the identification, measurement, and/or characterization of many tumor-associated particles in multiple bodily fluids. These include the measurement and analysis of circulating cells in blood, such as CTCs, circulating endothelial cells (CECs), circulating stem cells, and circulating stromal cells; the measurement of circulating nucleic acids, such as cell-free circulating tumor DNA (known as tumor-related cfDNA or ctDNA), circulating RNA, microRNA (miRNA), and long non-coding RNA (lncRNA); and measurement of RNA in tumor educated platelets (TEPs). Liquid biopsy also includes the investigation of tumor-associated extracellular vesicles (EVs), including exosomes, microvesicles, and large oncosomes. Other bodily fluids under study are urine, saliva, tears, cerebrospinal fluid (CSF), pleural fluid, and aqueous humor of the eye. In most cases, liquid biopsy is intended to facilitate non-invasive or minimally invasive serial sampling of cancer-related products in real-time during the initiation, treatment, and course of disease.

Currently explored clinical applications for liquid biopsy address the following needs:

• Monitoring for treatment response and the resistance to treatment in metastatic disease.

• Surveillance for micrometastatic disease following treatment of primary cancer (i.e., determination of residual disease or tumor recurrence after initial treatment of the cancer).

• Determining residual cancer burden following neoadjuvant treatment of a primary cancer, therapy that is given to shrink or ablate the tumor prior to its surgical removal and/or irradiation.

• Verification of cancer in high-risk or other individuals with imaging and/or test abnormalities such as abnormal mammograms, abnormal chest computed tomography (CT) scans or high prostate-specific antigen (PSA) levels in blood.

• Screening of asymptomatic patients for cancer.

Challenges to the field include a low signal-to-noise ratio (or “finding a needle in a haystack”), which results from the number of tumor cells or absolute quantity of tumor-specific nucleic acids or extracellular vesicles being lower by multiple orders of magnitude than other “normal” cells, nucleic acids, and extracellular vesicles in blood or plasma, making capture and analysis of tumor-specific cells or products a significant limitation to overcome. Second, normal cells or nucleic acids may contain the same surface markers or mutations as some cancer cells, especially as a person ages, which is when cancer is also more likely to occur. Third, there may be multiple subpopulations or phenotypes of cancer cells (e.g., multiple molecular subpopulations of single or clustered CTCs) or nucleic acids or extracellular vesicles that add further complexity. Finally, proof of clinical utility is critical: this means that a test must be shown to not only work as expected, but it must also change and improve clinical treatment and outcome compared to currently used strategies.

Because of the critical importance and fast pace advancement of the liquid biopsy field, we have proudly assembled this thematic collection of liquid biopsy articles from multiple noted authorities worldwide. This should serve as a point for understanding the state of the art and highlighting areas where the field is moving, so that together, we can advance the science and the clinical applications of these exciting technologies. Several of the publications included in this thematic collection focus on advanced methodologies to isolate particulate material from blood for diagnostic as well as therapeutic purposes. In the case of intact cells, some of the innovative approaches include label-free isolation of CTCs using a magnetic micropore chip, interfacial viscoelastic microfluidics, acoustophoresis, a size-tunable microvortex, and ferro-hydrodynamics. Each of these CTC isolation approaches provides different benefits and enables the field for new possibilities for the analysis and culture of CTCs. There are also several papers in the collection to explore new manufacturing approaches such as 3D printing, the isolation of CTCs covered with platelets as well as specialized microchips for capture of clusters of CTCs. Advanced methodologies are also reported for the isolation of cfDNA using microfluidic solid phase enrichment and exosomes using submicron focusing and microaffinity approaches.

As the field of liquid biopsy is rapidly maturing, there are now sophisticated approaches to perform liquid biopsy at the point of care, which is likely to impact the management of cancer patients in resource limited regions for global health. Another important innovation is on-chip and rapid analysis of the isolated biomarkers from blood, including in situ RNA analysis, sequencing of encapsulated single cells, use of magnetic forces to phenotype the expression level of tumor specific antigens on cells, microfluidic digital analysis of exosomes, as well as machine learning. One of the exciting possibilities recently emerging is the use of multi-analyte approaches to isolate and probe into cfDNA, exosomes, and/or CTCs as well as specific proteins from the same blood sample.

Liquid biopsy is taking a stronghold position in the monitoring, management, and ultimately screening of cancer patients, and the articles in this thematic collection provide an excellent perspective to the field not only for the newcomers but also for those who are active participants.

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