Widespread study of circulating tumor cells (CTCs) began in the early 2000s. Since then, the goal has been to replace expensive and invasive surgical biopsies with relatively simple and inexpensive CTC-based liquid biopsies. However, the translation of CTC-based liquid biopsy technology from research labs to the clinic has been moving forward slowly. A review published this year in Current Oncology Reports on the use of liquid biopsies in metastatic prostate cancer listed 15 platforms as examples of those used in research for CTC enrichment and detection. Yet, CellSearch, cleared by the FDA in 2008 for CTC enumeration, has been the only platform approved for clinical use in the past decade. And it is only approved for use in breast, prostate, and colorectal cancer patients.
Currently, physicians use CellSearch to count the number of CTCs in a 7.5-mL sample of blood. These counts have proven useful in prognosis and monitoring in the three types of cancer for which the technology is approved. In advanced prostate cancer, for example, 5 or more cells in a 7.5-mL sample has been shown to be associated with a worse prognosis. Physicians also use continuous counts of CTCs in monitoring the efficacy of a treatment. Though useful, this is far from fulfilling the promise of CTCs.
Researchers are hoping recent clinical findings will help move CTC-based liquid biopsies into the clinic more quickly. In addition to counting CTCs, they are now characterizing them and using that information to successfully make treatment decisions. Physician researchers are investigating the use of multi-parametric tests that will utilize both CTCs along with another blood-borne analyte—circulating tumor DNA (ctDNA). In addition, biotech companies, in collaboration with academic researchers, are working to introduce new, more sensitive CTC retrieval platforms—and get them FDA approved.
In a study published in July in JAMA Oncology, researchers from Memorial Sloan Kettering Cancer Center, the Royal Marsden Hospital in the U.K., and Lawson Health Research Institute in Canada showed that they could use a characteristic of CTCs to predict the optimal treatment for patients with advanced prostate cancer. The blinded, four-year study utilized the Oncotype DX AR-V7 Nucleus Detect test to determine if the nuclei from CTCs taken from 142 castration-resistant prostate cancer patients contained a protein called AR-V7. It is one of the first tests to validate the predictive value of a liquid biopsy for therapeutic response and demonstrated survival benefit. The researchers found that patients who had this variant present in the nucleus lived longer when treated with taxane-based chemotherapy, while those who tested negative for nucleic AR-V7 lived longer when treated with hormone-targeting therapy using androgen-receptor signaling (ARS) inhibitors.
“The importance of this treatment decision cannot be understated,” said Ryan Dittamore, chief of medical innovation and head of translational research partnerships for Epic Science, the company that offers the test for nucleic AR-V7 in partnership with Genomic Health. The test results are available to physicians in less than one week, an improvement over ctDNA tests that can take two to three weeks. Because these patients deteriorate rapidly, and weeks-long delays can be detrimental to the chances of survival, the days saved via the test can be vital in providing appropriate treatment, Dittamore explained. In the current study, researchers also validated the test across patient risks, to ensure the biomarker effect was due to the biomarker rather than other clinical variables. “We now have independent validation that the test is more predictive than physician intuition alone, meaning the test can help to extend the life of patients,” he concluded.
Meanwhile, the search is on for biomarkers like AR-V7 in all cancer types as scientists continue to make discoveries about the clinical significance of the number of CTCs present in a patient’s blood. Anthony Lucci, M.D., a professor of breast surgical oncology at MD Anderson Cancer Center, has shown that CTCs have prognostic value and that what scientists are learning in one type of cancer informs CTC-based work in others. In 2015, Lucci and his colleagues published a prospective study in the Journal of the National Cancer Institute that showed that the presence of even one CTC in patients with stage III inflammatory breast cancer—a rare and aggressive form of the disease—after primary chemotherapy were at high risk for relapse. In 2016, he and his colleagues showed in another prospective study that one or more CTCs at baseline were associated with progression within 180 days in stage IV melanoma patients. When it comes to CTCs, the two types of cancer are more similar than one might think. “Blood-based detection of CTCs in melanoma works even better than we thought,” he noted.
Lucci said he is looking forward to the results of ongoing clinical trials, like the DETECT study, currently underway in Europe, that is looking at the possibility of using HER2 expression in CTCs to guide treatment decisions. In the meantime, Lucci and his colleagues are looking at the use of CTC counts from sequential blood draws in order to determine what these counts reveal about which breast cancer and melanoma patients will respond to treatment. “We have such a large volume of patients that we can realistically accomplish this in the near future,” he said.
CTCs vs. ctDNA
Despite these kinds of advancements, the truth is that CTCs are an incredibly rare component of a cancer patient’s blood. A significant amount of research has shifted away from CTCs to circulating tumor DNA (ctDNA) thanks to improvements in the depth, accuracy, and cost of high-throughput, next-generation sequencing technology. “You get to see just about every piece of DNA you had in your tube,” said Amir Goldkorn, M.D., a prostate cancer researcher and associate professor of medicine at the University of Southern California’s Keck School of Medicine (USC). Goldkorn was one of the authors of the 2016 Current Oncology Reports review on liquid biopsies in prostate cancer. “Now that you have this technology, some investigators prefer to avoid the complexities and difficulties of isolating the actual cancer cells, because analyzing the cell-free DNA can be technically more straightforward.”
However, there are compelling reasons to continue to study CTCs, said Goldkorn. CTCs still represent the entire content of the cancer cells that make up the primary tumor and metastases. “DNA is just one part of the profile, whereas CTCs are living cells that contain the entire complement of DNA, RNA, and protein.” By definition, ctDNA is only a subset of tumor DNA, DNA fragments that were released from dying cells. The bottom line, Goldkorn said, is that researchers have yet to complete the kind of large, prospective, well-controlled studies needed to determine which approach, CTC-based technologies or ctDNA-based ones, best informs treatment decisions and improves clinical outcomes. “That’s the kind of work that needs to be done.”
That’s exactly the kind of work Goldkorn is doing. He said the answer may be a multi-parametric approach to liquid biopsies that attempts to get various kinds of information from a single blood sample. “We want to see what we are learning from each type of data, to what degree they overlap, and to what degree they provide unique information. We want to understand how to use these technologies to develop the best predictive and prognostic tools that we can use for our patients in a way that is efficient, inexpensive, and non-invasive.” Goldkorn said his group is conducting pilot studies, in addition to large, prospective, multi-center trials.
The goal for multi-parametric studies is to take different technologies, apply them in an integrated manner to a single sample of blood, and develop the workflows where results can be viewed in parallel. “What we have seen so far is that you get complimentary information. It’s important to look at both CTCs and ctDNA,” Goldkorn said. Researchers are also looking at RNA- and protein-based tests, as well as studying extracellular vesicles. Once these studies are done, they will lay the foundation for commercial assays. “We may find we need the CTCs because we want the counts and to look for (protein) variants, but we want the cell-free DNA data because they give us the best profile of tumor mutations.” But Daniel Danila, M.D., is skeptical about the future of multi-parametric tests. “It’s theoretically a great idea. Practically, we have a lot of research to go. Right now, the buffers you use in these tests are very different,” said Danila, a medical oncologist at Memorial Sloan Kettering Cancer Center.
Increasing Detection Sensitivity
Danila’s work focuses on improving CTC detection. To do that, many are investigating new ways of harvesting CTCs. In 2016, Danila and his colleagues published a paper in The Cancer Journal that showed that two detection methods, AdnaTest Assay and ddPCR analysis, both detected cancer cells in spiked samples of blood more often than CellSearch. AdnaTest correctly found CTCs in 62% of the samples, ddPCR detected cells in 69% and CellSearch had a 45% detection rate. “The ddPCR assay required the lowest blood volume, least on-site processing, and longest stability for batch processing,” the researchers wrote. “We need increased sensitivity. It’s hard to monitor our patients if we can’t detect CTCs, which serve as surrogate markers for survival in many cancer types.”
Menarini Silicon Biosystems, the current makers of CellSearch, is working on further developing its system, said Nicolò Manaresi, Ph.D., the company’s chief scientific officer. “CellSearch has a leading role in the field of CTC. This is because of the clear definition of a context of use, standardized reagents and degree of automation which many of the alternative technologies for CTC enrichment and enumeration still lack,” Manaresi said. However, the company is not standing still. “We keep working on further developing the system.” Currently, the company is expanding the menu of assays that can be run on the CellSearch platform. It is also investing in the integration of CellSearch with DEPArray and Ampli1 technologies to deliver sample to result workflow which can provide molecular characterization of CTCs with single-cell resolution. “Clearly, evaluation of genomic biomarkers is a hot topic in liquid biopsy,” Manaresi said. The workflow is intended to meet this need.
While there is much work being done to achieve the potential of circulating tumor cells (CTCs) for molecular and other analysis, capturing and harvesting them without damaging the cells remains a challenge. One company that is developing a solution to this challenge is ANGLE plc. The company’s Parsortix microfluidic system—for which is currently seeking FDA clearance—captures CTCs of all types. Unlike CellSearch technology, which is based on the binding of epithelial cell adhesion molecules (EpCAM) and does not capture all CTCs because they don’t express the necessary cell surface markers, the Parsortix design enables the capture and harvest of virtually all CTCs in a blood sample.
The system utilizes a 10-mL sample of blood, which is passed through a Parsortix separation cassette. The user removes the top of the 10-mL tube of blood and attaches it to the Parsortix instrument, which then pushes the blood through a single-use cassette the size of a microscope slide. The larger, less compressible CTCs are gently held within the cassette, while the blood cells pass through the cassette to waste. Once the separation process is complete, the user can either automatically stain the captured cells with various antibodies within the cassette for examination, or they can instruct the system to harvest the cells out of the system for molecular and other analysis.
“There are many things you can do with CTCs if you can get them out of a sample without damaging them,” said Andrew Newland, ANGLE’s founder and CEO. “Our system captures all of the different phenotypes of CTCs in the blood. The antibody-based systems don’t capture the mesenchymal cells which are the cause of secondary cancer.”
Several leading investigators are currently using Parsortix in their CTC research. Danila serves as a scientific advisor to ANGLE and uses the platform in his work. Goldkorn, and ANGLE customer, agrees CTCs are valuable for research. “There are many clinical trials aimed at developing robust and analytically validated assays to identify, enrich, an characterize CTCs.” As to whether this work will result in better or complimentary information compared to that gathered form ctDNA, Goldkorn said, “The jury is still out.”