Circulating tumor cells (CTCs) are, by definition, epithelial cells with an intact nucleus that express cytokeratin and don’t express the leukocyte-common antigen CD45, which are found in the blood of cancer patients.
Their presence and abundance in patients who have undergone treatment has been shown to correlate with disease progression and overall survival. They are extremely rare even in such patients: estimates are in the range of 10-8–10-9, and 50 from a milliliter of blood is considered a good yield.
Yet no one truly knows what CTCs are, where they come from, what they mean, what the best way is to get at them, or even what to with them afterward. There is a single FDA-sanctioned technology, Veridex’ CellSearch, approved only for the semi-automated enumeration of CTCs from 7.5 mL of whole blood.
That doesn’t stop other techniques—some of which build upon the CellSearch platform, others utilizing distinct technologies—from generating a lot of excitement as well. Academic scientists, physician-researchers, and industry players gathered at Select Biosciences’ inaugural “Circulating Tumor Cells” conference to discuss how to capture, analyze, and interpret the data from these putative biomarkers that many feel have the potential to significantly impact clinical oncology.
Minetta Liu, M.D., associate professor at Georgetown University Medical Center, uses CellSearch for “all of my patients within the indication of metastatic disease, and I have found it useful in timing imaging studies, in helping to determine in conjunction with routine clinical factors and our radiological studies whether current therapy is helping a patient or not,” she said. “We don’t know what we’re counting, but I do know that counting the cells with that technology does correlate with patient outcomes.”
The hope is to go beyond simply enumerating CTCs. “We need to figure out what these cells are so we can manipulate them, to do better in those outcomes,” she added. Dr. Liu is involved in a clinical trial comparing molecular features of CTCs with those from primary breast tumor or metastatic sites, “trying to get an understanding of what these cells are trying to tell us.”
Stefanie Jeffrey, M.D., chief of surgical oncology research at Stanford University School of Medicine, is interested in how CTCs may be used to individualize therapy. Even from the same blood draw you can see different types of CTCs, and this may have implications for treatment, especially in a metastatic setting. One drug may be needed to treat one kind of metastasis and another drug to treat another, but you usually cannot biopsy all metastases a patient has.
“If you could do a liquid biopsy in real time, and could see how the tumor cells are changing, then potentially you may be able to more intelligently select the therapy—but this would have to be proven within a prospective clinical trial,” Dr. Jeffrey said. “We’re hoping that CTCs that are released to the bloodstream may be a surrogate, representing some of the cells that are involved in the seeding and reseeding of metastases.”
Lihua Wang, M.D., Ph.D., senior scientist of laboratory of toxicology and pharmacology at Frederick National Lab, develops CTC assays for targets that have been demonstrated for tumors, to have a way to measure the effect on tumor cells multiple times after administration of a drug—the assumption being that what’s happening in the CTCs is accurately reporting what’s happening in the tumor. But this needs to be proven. In fact, one of Dr. Wang’s fundamental goals is to establish whether a CTC is an equivalent to a biopsy.
A true surrogate not only accurately reports the same effect on its target, but reports it quantitatively as well. For the assays they have looked at there is concordance, she noted. “We have not yet proven that it’s a surrogate. That’s a high hurdle.”
Cells can be captured part way through the automated steps of CellSearch for the purpose of characterization, and there are also other ways to collect CTCs as well. These may not capture the same set of CTCs, and “it’s important to look at these side by side and to try to really figure out what they are and where they might be most useful,” remarked Dr. Liu.
Among these is the MagSweeper, an automated device that uses a reiterative process of magnetic capture, wash, and release to enrich CTCs by up to 108, yielding a highly purified sample of live CTCs that can be analyzed on a single-cell basis. The MagSweeper was invented by Dr. Jeffrey and Stanford colleagues from the Genome Technology Center and School of Engineering, and has been licensed to Illumina.
Dr. Jeffrey donates her royalties to a nonprofit charity “so that I can use other technologies that may isolate more CTCs but maybe not as pure for different purposes, or to capture different phenotypes of CTCs,” she pointed out. “And so we’re testing other technologies in our lab as well.”
The On-Q-ity platform combines affinity capture with size filtration capture to isolate CTCs. About 100,000 micropillars, conjugated with antibody, line the chamber of the plastic microfluidic device. The micropillars are arrayed in a gradient—they “get closer and closer together as the blood flows through the device, allowing you to capture the cells based on size in addition to affinity,” explained Kam Sprott, Ph.D., the company’s director of product development. The smaller leukocytes should be able to flow through even the smallest gaps, while CTCs are retained in the chamber for imaging or further processing.
Another way to achieve a similar end is to “make sure that cancer cells collide with the walls of the chip very often, but the contaminating cells—the things we don’t want to capture, which is mostly white blood cells—don’t collide with the surface anywhere near as much,” said Brian Kirby, Ph.D., who directs the Micro/Nanofluidics Laboratory in Cornell University’s Sibley School of Mechanical and Aerospace Engineering. He used his training in fluid mechanics to design a three-dimensional geometrically enhanced differential immunocapture (GEDI) microfluidic device in close collaboration with Weill Cornell Medical College’s David Nanus, M.D., and Evi Giannakakou, Ph.D.