Understanding Circulating Tumor Cell-Based Liquid Biopsy
Our blood is a remarkable substance. Medically, blood is considered a connective tissue, and the passage of blood through the circulatory system connects every organ system and living cell in the body. Because cells continually shed biomolecules into circulation, a sample of blood is a rich snapshot of state of the body at any given moment in both health and disease. Liquid biopsy assays for cancer detection and treatment are being developed to harness that information for medical use and improve outcomes for each individual patient. In this article, we explore the identification, characterization, analysis, and clinical applications of circulating tumor cells as part of blood-based liquid biopsy for cancer management.
What are circulating tumor cells (CTCs), and how are they detected?
As tumors grow, they can attempt to expand to new sites of the body by “seeding” cancerous cells into circulation. The cells that survive their passage through the blood can take root in distant sites to initiate metastasis, the most lethal form of the disease. Although extremely rare (usually detected at a rate of a few cells per milliliter of blood, or one in a billion circulating cells), these circulating tumor cells can be detected in blood samples based on their cell-surface or intracellular markers as well as physical properties. Direct imaging approaches utilize fluorescent labeling and imaging followed by advanced computational methods to identify CTCs. Examples of clinical research platforms using this approach include the University of Southern California’s High Definition Single Cell Assay (HDSCA) Discovery Platform, RareCyte’s CyteFinder platform, and Epic Sciences’ Comprehensive Cancer Profiling platform.
Liquid biopsy can be used to isolate circulating tumor cells from the bloodstream. Figure created with Biorender.
CTCs captured by cell-surface markers can be isolated through positive selection techniques such as fluorescence or magnetic-activated cell sorting (FACS or MACS). CellSearch (Menarini) is based on positive-selection MACS and was the first FDA-approved CTC capture platform. CellSearch is currently approved for use in metastatic breast, prostate, and colorectal cancer for disease monitoring. Negative depletion, which uses surface markers and other properties to deplete all non-CTC cells, is an alternative approach. CTCs can also be separated from non-cancerous cells based on their physical properties (e.g. larger size). Parsortix (ANGLE), uses a microfluidics system to isolate CTCs based on size and deformability, and is FDA-cleared for use in enrichment of CTCs in metastatic breast cancer. For more technical detail on CTC capture and analysis, take a look at this review.
A major advantage of isolating whole cells from the blood is the full complement of biomolecular and functional information that comes along within each cell: the entire genome, transcriptome, epigenome, and proteome can be analyzed, and CTCs can be used for downstream functional studies assessing characteristics such as therapeutic response. Single-cell multi-omics on CTCs has the potential to provide a more complete picture of tumor heterogeneity and the molecular landscape of a given cancer, informing patient treatment decisions as well as potentiating basic research on the biology of cancer metastasis. For more on the many multi-omics research studies examining CTCs, see this review.
Circulating tumor cell isolation techniques. Figure adapted from Ju et al. (Biomarker Research) 2022.
How are CTC-based liquid biopsy assays used clinically?
CTC profiling is largely in the early stages of clinical use for most cancer patients. However, this technology has reached clinical utility in prostate and breast cancer and several tests are available to guide treatment decision and prognosis.
One of the most well-developed and clinically supported applications of CTCs is in prostate cancer. The presence of AR-V7, a splice variant of the androgen receptor that acts as an oncogenic driver, has been demonstrated to predict poor outcome in patients undergoing endocrine therapy. Qiagen’s AdnaTest AR-V7 assay (for research use only) and Epic Sciences’ Oncotype DX AR-V7 Nucleus Detect assay have been developed for assessment of AR-V7 status in CTCs on the basis of this work, and clinical trials are ongoing. AR-V7 status is used to guide treatment decisions, and Epic Sciences’ test is currently covered by Medicare. An additional application of CTCs with demonstrated clinical utility in the management of prostate cancer is the use of CTC count/enumeration—the number of cells detected in a given blood sample—as a surrogate biomarker for treatment response, risk of relapse and overall survival.
CTC count and molecular profiling assays have been assessed in the management of breast cancer and have prognostic and cancer recurrence predictive power. Several studies have demonstrated that CTC count is correlated with overall survival and progression-free survival in breast cancer patients, and many clinical trials on ongoing in this space. There is also ongoing research on the utility of determining the presence of HER2, a marker used to guide therapy selection in breast cancer, and PD-L1, an immunotherapy biomarker, on CTCs. A more comprehensive list of clinical trials assessing clinical applications of CTCs across cancer types can be found in this review.
What are major barriers to clinical application of CTC-based assays?
One significant impediment to the use of CTC profiling across a broad range of cancers is the great heterogeneity of these cells: the field does not yet have a solid understanding of what subtypes of CTCs lead to metastasis, or what CTC cell surface markers are indicative of cancer progression. To prevent overtreating and unnecessary intervention, these critical knowledge gaps must be met to allow the development of clinical assays for a broad range of cancer types.
In addition, many studies have identified insufficient sensitivity of existing CTC counting platforms as another major hindrance for the progress of this analyte in routine clinical care. When decision-making and risk stratification is based on single-digit counts, a very high bar is necessary to avoid false negatives. However, despite their relative scarcity in a standard peripheral blood draw, the richness of biological information contained within CTCs continues to drive research on improved isolation and analysis approaches, including large scale sampling and streamlined approaches to downstream analyses. Multimodal liquid biopsy approaches, combining CTC enrichment and analysis with other analytes such as serum biomarkers, circulating tumor DNA or oncosomes (large extracellular vesicles released by cancer cells), are at the forefront of research in this field and will continue to expand the actionable information that can be derived from a single blood sample.
