Photos courtesy of Mehmet Toner and Bill Truslow Photography www.truslowphoto.com.
In 2007, a Harvard-based research team changed the face both of microfluidics and cancer detection forever when they described in Nature their novel CTC (circulating tumor cell) chip.
CTCs are cells that spread from primary solid tumors through the bloodstream to the bone, brain, lungs or liver. The cells are very rare—about one in a billion—and were virtually undetectable in blood until the advent of the chip.
The CTC chip has since gone through several iterations, but originally it contained about hundreds of thousands of micropillars coated with antibodies, which bind to CTCs in a blood sample without binding normal blood cells. Antibody binding renders the CTCs detectable and ready for enumeration and molecular extraction. The authors reported that the chip isolated CTCs from the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer and in those with early-stage prostate cancer with a high degree of sensitivity and specificity.
The Nature paper was quickly followed by an article in the New England Journal of Medicine that served as a clinical proof-of-principle for the new technology. The researchers used the chip to capture CTCs from the blood of patients with non-small-cell lung cancer. In about half the patients, they detected the T790M mutation, which confers resistance in those with EGFR mutations who receive treatment with tyrosine kinase inhibitors, and is associated with reduced progression-free survival. The authors concluded at that point that CTC chip technology could eventually be used to monitor the emergence of resistance during the course of cancer treatment. More recent work involved various improvements on the technology published in PNAS (2010), as well as exploring the biology and the clinical utility of CTCs in a subsequent paper published in Nature (2012) to show a critical role for wnt2 signaling in pancreatic cancer metastasis, and in the November 2012 issue of Cancer Discovery to measure treatment-induced androgen receptor signaling in prostate cancer patients.
The research team that made these and related seminal discoveries was led by SLAS2013 keynote speaker Mehmet Toner, Ph.D., the Helen Andrus Benedict Professor of Biomedical Engineering at the Massachusetts General Hospital, Harvard Medical School and Harvard-MIT Division of Health Sciences and Technology. Toner also is founding director of the NIH BioMEMS [BioMicroElectroMechanical Systems] Resource Center, which fosters the development of living-cell-based microdevices that enable biomedical investigators "to probe, manipulate, engineer, and analyze biological cells for diagnostic, prognostic and therapeutic advances and basic biology discovery."
SLAS2013 Program Committee Co-Chair Aaron Wheeler, Ph.D., University of Toronto, says, "I am really, really excited about having Mehmet Toner speak at the conference. His work with CTCs has rapidly spawned a whole subdiscipline of microfluidics. His patents in this area were invested in by Johnson & Johnson and there is a huge amount of excitement in the scientific and medical communities for big results soon. I can't wait for his presentation."
Toner's talk, "Bioengineering and Clinical Applications of the Circulating Tumor Cell Microchip," is one of two sessions that will be live-streamed during the conference on January 14, 9:00 a.m. U.S. Eastern time.
"Although a blood test is the most commonly used diagnostic test in medicine, normally it is useful only when there are large numbers of the sought-after cells in the sample," Toner says. "But for serious diseases, the much rarer cells, such as CTCs, really hold the key to early diagnosis, treatment management and patient monitoring."
Until Toner's team began their work, the laboratory automation and sample processing technologies needed to routinely isolate these rare cells were not readily available. "You cannot just look into a finger prick of blood—you're not going to find a rare cell in such a tiny sample," Toner explains.
Ironically, at the time, "the field of microfabrication was going in the direction of managing and processing smaller and smaller amounts of bodily fluids for varied diagnostic purposes. But nobody was thinking about miniaturization or microfluidics as a way to process large volumes—10 or 20 milliliters—of whole blood," Toner says. He determined that the same technologies that were being used to gather information at the single-cell level, enabling the researchers to reduce the sample size needed for processing, could also be used to process larger samples while retaining single-cell level control.
"We turned the field upside down and essentially created a whole new discipline," Toner states. "Until our first papers, the notion was that the immune system removed most CTCs from circulation, and that you could find them only in a small fraction of even metastatic patients. Therefore, while they might be useful for prognosis, they could not provide clinically actionable information.
"We challenged that notion. We said, ‘No—the cells haven't been removed from circulation. The technologies simply aren't sensitive enough to detect them,'" Toner continues. "In our first paper we showed that while CTCs are rare compared with other cells, they are much more common than originally thought." Toner describes the moment as "poking a sleeping bear." The result was a "wave of interest in the field" that has continued to mushroom. Today, CTCs are the subject of several world congresses and the U.S. National Institutes of Health has created a CTC study section.
Because CTC chip analysis is minimally invasive compared with a solid-tumor biopsy, and also is likely to be less expensive and easier on patients, Toner anticipates several near- and longer-term applications for the clinic. "The most exciting near-term applications will be in targeted therapy for cancers of the lung, breast and skin for which we already have molecular markers," he says.
The 10-milliliter blood sample drawn from a patient would serve as a "liquid biopsy," Toner explains. "We would extract the CTCs from the blood, sequence them and look to see if they contain genetic markers that could affect treatment." For example, the team recently found that gastric adenocarcinomas resulting from amplification of the growth-factor receptor gene c-MET respond only to novel inhibitors of the MET tyrosine kinase. This has led to the initiation of a genotype-directed clinical trial.
Toner notes that such discoveries were not possible until recently because some biomarkers—such as the ALK [anaplastic lymphoma kinase] translocation found in non-small-cell lung cancer and other malignancies—can be obtained only from RNA, not free DNA floating in blood—and RNA can be analyzed only from live cells. "Many of the earlier technologies for fixing and processing cells were so harsh from a lab automation perspective that cells couldn't survive. But we need live, intact cells to obtain high quality information that might be clinically useful." With newer processing techniques, most cells survive, enabling further investigation. To date, the chip has identified more than 1,200 cancer-causing genetic mutations.
Perhaps because traditional cancer biopsies are invasive and debilitating to patients, often they are done only once. "Yet most patients develop resistance to therapy over time, and the genetic status of their cells needs to monitored longitudinally. A non-invasive test like ours would enable clinicians to personalize targeted therapies and monitor treatment response over time," Toner observes. Clinicians could first determine a first-line treatment, then switch to a second- or third-line treatment as soon as any resistive mutations occur.
CTC chip technology also would enable clinicians to monitor cancer patients in a manner similar to the way AIDS patients are monitored. "In AIDS, you would be monitoring viral load and deciding a course of action. In cancer, CTCs, or ‘tumor load,' would be monitored," Toner explains. A large number of tumor cells in blood would probably indicate that the patient is not responding to treatment. "You could also monitor longitudinally to determine if a patient is in remission or if there is a recurrence. You can't do biopsies or expose patients to radiation in imaging tests on a monthly basis," he says. "But with the CTC chip, you could follow a patient more often and more closely and identify recurrence or resistance early on."
In the longer term, the goal would be to use the technology for early detection. "Those kinds of studies will involve much larger clinical trials and, for the technology, a very high degree of sensitivity and specificity," Toner acknowledges. "That's because if you look at a million people, even a tiny amount of false positives will scare a lot of individuals." Such trials also would require large-scale funding. "But that would be the holy grail—where we could isolate CTC cells early, and monitor certain mutations over time, particularly for cancers we know tend to become aggressive."
Eventually, "in the very long term," CTC technology might be used to screen the general population, Toner suggests. "The screening could be included in the blood analysis when you go for an annual checkup with a primary care physician, just like a CBC [complete blood count]."
Generally, in clinical research, "we know the clinical endpoint, but there's no technology to get there," Toner observes. "Ours is the opposite—we're developing the technology, but we still need to figure out the right clinical applications at each stage." Adding to the challenge is the fact that because CTCs have only recently become available for laboratory studies, "we need to get a much better understanding of the biology of these cells."
Massachusetts General Hospital is a "unique environment" that enables bioengineers, molecular biologists and clinicians to work together, setting the stage for some "major clinical breakthroughs," Toner adds. With respect to CTC development, "we are trying to do all three at the same time—hone the technology, better understand the biology of the cells and test clinical applications. Sometimes the technology is behind, sometimes the biology is behind—it's truly an evolving process, and the innovation and creativity required can only be found in a truly integrated, multidisciplinary environment."
The team also is working with Johnson & Johnson on a newer version of the technology. Toner anticipates that a paper on the "next-generation" CTC chip, which should enable "much earlier detection," will be published by the time he presents at SLAS2013.
What kinds of opportunities does the emerging field of CTC research offer scientists and researchers? "Given that almost every pharmaceutical company is working on some way of isolating circulating tumor cells, my prediction is that over the next five to 10 years, CTCs will become a true clinical reality," Toner affirms. "When that time comes, knowing how to look at these cells and make clinical judgments would become a very important set of skills."
In cancer, for example, the possibilities go beyond determining responsiveness to targeted cancer drugs. CTCs also offer the opportunity to study "cancer stem cells," or "metastasis precursors"—thought to be at the origin of cancer spread via the bloodstream—to define their molecular vulnerabilities and help design new therapies to prevent cancer from spreading.
In addition, "the whole field of pathology is changing in dramatic ways," Toner says. "For one thing, there is much greater use and need for digital tools. But also, several relatively new vaccinations are making certain conditions, such as cervical cancer, less common, and so there is less need for some of the cytopathology expertise we had in the past." Instead, pathologists will need to use their skills to assess CTCs for use in diagnostics and decision making—"and experts who can do so will be tremendously valuable."
Toner adds that the opportunities extend beyond CTCs to other rare cells. "Fetal cells and other cells in maternal circulation would be a huge area for prenatal diagnosis," he suggests. "Another exciting application would be in infectious diseases such as tuberculosis, where analysis of rare circulating cells might enable diagnosis of latent disease," he says. "Dendritic cells and immune cells also will require automated point-of-care processing, and we'll need to do it quickly, without altering physiology or biology, so we end up with cells that are still clinically useful."
The bottom line is that "this kind of technology will create a very powerful bridge that might ultimately enable us to diagnose almost every single disease, including Alzheimer's, in blood," Toner says. "Right now, we don't know how to find the rare particles that will make this a reality. They could be exosomes, they could be DNA, they could be cells. But this is the direction in which medicine is going—noninvasive methods to get higher quality information from blood and other bodily fluids. CTCs are one big, but early, example. There will be many more to come."
October 26, 2012