Contributed Commentary by Candice Willey
June 3, 2020 | When cancer is diagnosed at an early stage, before metastasis, patients typically experience significantly higher rates of survival. In metastasis, however, those chances decrease greatly when circulating tumor cells (CTCs) break away from the primary cancer, travel through the bloodstream and form new tumors in other parts of the body.
Detecting CTCs in the blood can offer advantages over the detection of other substances, such as circulating tumor DNA, where dead cells get broken down and their contents—including DNA—are released into the bloodstream. In fact, analyzing cells in their entirety can reveal a number of insights about a tumor, such as whether it expresses a targetable receptor and the degree of tumor heterogeneity. CTC-based liquid biopsies could be used for tracking, monitoring, and predicting the efficacy of therapy and emerging resistance.
Yet, despite the benefits, the potential of CTCs to serve as a liquid biopsy for cancer has been underutilized, mostly due to the inability to acquire intact CTCs, along with the associated technical challenges. More importantly, until now, the role of CTC information in the design of oncology clinical trials has been greatly overlooked.
In this article we will discuss (1) the difficulties of using CTCs in diagnostic testing despite their promise, (2) the emergence of new CTC technologies to improve tumor sample accessibility, along with the advantages and disadvantages of each approach, and (3) the benefits of integrating CTC information into clinical trial decisions.
Challenges of using CTCs for diagnostic testing
One of the greatest challenges of using CTCs is their rarity in blood samples, with one milliliter of whole blood acquired from a cancer patient having anywhere from one to 10 CTCs among millions of blood cells. This makes designing CTC-based diagnostics difficult because it requires a test that is highly sensitive. In addition, CTCs have a short half life, which makes achieving a high yield and purity difficult.
Further, in immuno-oncology trials, there is often high variability among patient samples and assays. CTC analysis is designed to detect rare events, and readouts may vary for any given test. As a consequence, CTC assays have to be performed more frequently during the life of a clinical trial to minimize this variability. And finally, historically, a majority of analyses have been limited to bulk-cell strategies, missing the opportunity to acquire information on cellular heterogeneity.
While challenges in using CTCs remain, new single-cell technologies—also known as CTC assays—are emerging that can efficiently capture and characterize these cells.
Advantages and limitations of new single-cell technologies
Today, the development of powerful single-cell technologies have made it possible for researchers to probe the genetic material of CTCs at the single-cell resolution, allowing for the study of spatial and temporal dynamics. These assays—which directly measure the concentration of CTCs, and provide a finer-grained picture of complex biology and unmask heterogeneity that is present in tissues—can improve tumor sample accessibility and reveal molecular profiles of tumors for more informed targeting and prognosis.
At the same time, however, each of the current technologies has its own unique issues and challenges:
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Reverse transcriptase polymerase chain reaction (RT-PCR) — Higher sensitivity than immunocytochemistry, but often has false positives produced by nucleic acid contaminations
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Immunofluorescence and immunocytochemistry — Isolation of biomarker-positive CTCs, but possible loss of cells with lower levels of expression
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Fluorescence in situ hybridization (FISH) — Uses optical imaging system for enhanced sensitivity, yet cells are not viable after analysis
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Subtraction enrichment with immunostaining-FISH (SE-iFISH) — Provides characterization of each CTC subtype in the progression of the clinical course of the disease, yet cells are not viable after analysis
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Fluorescence-activated cell sorting (FACS) — Versatile technique that allows for several parameters to be changed simultaneously, but has limited throughput as cells are analyzed individually and flow sorting may be detrimental to cells
Benefits of integrating CTC assays into clinical trial decisions
Monitoring CTC genomic and epigenomic profiles has the potential to better inform a clinician’s treatment selection for a patient and their potential survival rate. Using globally standardized equipment and a single operational procedure for sample collection and slide preparation for nucleated cells can allow for quick, efficient, and highly-reproducible separation of nucleated cells from whole blood, and can generate uniform slide smears. The major advantage of this technique is the ability to process samples at regional labs, and store the prepared slides in the correct temperature prior to analysis.
Once analyzed, CTC information can be integrated into clinical decisions. In adaptive clinical trials, for example, CTC information could be used as an early indicator for drug candidate efficacy, especially when this information has coalesced into a full mutation profile for the tumor. By measuring CTCs early, a developer can cease a trial as soon as it is revealed that a drug is not efficacious, therefore saving millions of dollars in trial costs.
As drug development becomes increasingly expensive, sponsors will become more interested in accruing the best available data for trial planning and decision-making. Consequently, we are moving towards a future where all oncology clinical trials will include CTC assays because of their value in generating prognoses based on the effectiveness of a drug. Techniques will advance, and we will witness these assays being further integrated with next-generation sequencing and big data analytics.
Candice Willey is a Scientific Affairs Manager at ICON with more than 20 years’ experience in translational research and diagnostic assay development. She has experience in evaluating new technology platforms for RNA and DNA analysis and biomarker strategy design and implementation. She can be reached at Candice.willey@iconplc.com.