19 May 2016
Embedded in a small translucent wafer measuring just under an inch a side, the spiraling coils—like neatly packed iPod earbuds—aren’t much to look at.
But judging on appearance alone would sell short the brainchild of Chwee Teck Lim of National University Singapore and Jongyoon Han of the Massachusetts Institute of Technology. Those coils sift through millions upon millions of blood cells for faintly detectable indicators of a solid tumor lurking in a patient’s body—the handful of cancer cells that are often found circulating in the blood. Called circulating tumor cells, these cells may well be the seeds of distant metastases, which are responsible for 90% of all cancer deaths.
Over the past several years, researchers and clinicians have become increasingly fixated on these circulating cells as cellular canaries-in-the-coalmine, indicators of distant disease. The blood of cancer patients is chock-full of potentially telling molecules, and researchers and clinicians are hotly investigating these materials for their efficacy as indicators and predictors of illness, disease progression, response to treatment, and even relapse.
Soon, a simple blood test could reveal whether a person has cancer.
For patients with cancer, such tests could provide a welcome respite from painful, invasive, and sometimes dangerous biopsies that typically are used to track and diagnose disease—a fact reflected in the terminology often used to describe the new assays: liquid biopsy. For researchers and clinicians, they provide a noninvasive and repeatable way to monitor how a disease changes over time, even in cases when the tumor itself is inaccessible.
And unlike the finger-stick testing used by the embattled company Theranos, which recently voided two years of results from their proprietary blood-testing machines, the liquid biopsy methods being researched and developed by teams of scientists around the world use standard blood-drawing techniques and have been subject to peer review.
In the short term, researchers hope to use liquid biopsies to monitor tumor relapse, track a tumor’s response to targeted therapies, and match patients with the treatments most likely to be effective—the very essence of “personalized medicine.” But longer term, some envision tapping the blood for early diagnosis to catch tumors long before symptoms start, the time when they’re most responsive to treatment.
For now, most such promises are just that: promises. With the exception of one FDA-approved test, a handful of lab-developed diagnostics, and a slew of clinical trials, few cancer patients today are benefitting from liquid biopsies. But many are betting they soon will be. Liquid biopsies, says Daniel Haber, director of the Massachusetts General Hospital (MGH) Cancer Center, “currently are aspirational—they don’t yet exist in that they’re not part of routine care. But they have the possibility to become so.”
Despite its name, liquid biopsies are not exactly an alternative to solid tissue biopsies, says Mehmet Toner, a professor of biomedical engineering at MGH who studies circulating tumor cells. Patients who are first diagnosed with cancer via a liquid biopsy would likely still undergo a tissue biopsy, both in order to confirm a diagnosis and to guide treatment.
But liquid biopsies do provide molecular intel that might otherwise be impossible to obtain—for instance, in the treatment of metastatic disease. Oncologists typically biopsy patients with metastatic disease only once, to confirm the diagnosis, says Keith Flaherty, director of the Henri and Belinda Termeer Center for Targeted Therapies at the MGH Cancer Center. But such a test reveals the genetics of the cancer only at the sampled site. Many patients harbor multiple metastases, some in relatively inaccessible locations like the lungs, brain, or bones, and each may contain cells with different genetic signatures and drug susceptibilities. “Liquid biopsies provide an aggregate assessment of a cancer population,” he says.
Today, says Max Diehn, an assistant professor of radiation oncology at the Stanford University School of Medicine, oncologists can get a read on how a patient responds to therapy using a handful of protein biomarkers found in blood, urine, or other biofluids, such as prostate-specific antigen (PSA) in the case of prostate cancer, or using noninvasive imaging technologies like magnetic resonance imaging (MRI) or computed tomography (CT). But those tests often fall short. Many biomarkers aren’t specific enough to be useful, and imaging is relatively expensive and insensitive. Also, not everything that appears to be a tumor on a scan actually is. And, Flaherty notes, imaging studies reveal little or no molecular information about the tumor itself, information that’s useful in guiding the treatment.
In contrast, liquid biopsies can reveal not only whether patients are responding to treatment, but also catch game-changing genetic alterations in real time. In one recent study, Nicholas Turner of the Institute for Cancer Research in London and his colleagues examined cell-free tumor DNA (ctDNA), or tumor DNA that’s floating free in the bloodstream, in women with metastatic breast cancer. They were looking for for the presence of mutations in the estrogen receptor gene, ESR1. Breast cancer patients previously treated with so-called aromatase inhibitors often develop ESR1 mutations that render their tumors resistant to two potential treatments, hormonal therapies that target the estrogen receptor and further use of aromatase inhibitors that block the production of estrogen. Turner’s team detected ESR1 mutant ctDNA in 18 of 171 women tested (10.5%), and those women’s tumors tended to progress more rapidly when treated with aromatase inhibitors than did women who lacked such mutations. Those findings had no impact on the patients in the study—the women were analyzed retrospectively—but they suggest that prospective use of ctDNA analysis might be used to shift treatment toward different therapeutic strategies.
Viktor Adalsteinsson of the Broad Institute of MIT and Harvard, whose group has sequenced more than a thousand liquid biopsy genomes, calls the ESR1 study “promising and illuminating.” At the moment, he says, such data are not being actively used to influence patient treatment, at least not in the Boston area. But Jesse Boehm, associate director of the Broad Cancer Program, says he thinks it could take as little as two years for that to change. “I’ve been here at the Broad for ten years, and I don’t think I’ve ever seen another project grow from scientific concept to potentially game-changing so quickly,” he says.
Liquid biopsies generally come in one of three forms. One, ctDNA—Adalsteinsson’s material of choice—is the easiest to study, but also the most limited as it relies on probing short snippets of DNA in the bloodstream for a collection of known mutations. The blood is full of DNA, as all cells jettison their nuclear material when they die, so researchers must identify those fragments that are specifically diagnostic of disease. While the genetic mutations behind some prominent cancers have been identified, many more have not. Also, not all genetic changes are revealed in the DNA itself, says Klaus Pantel, director of the Institute of Tumor Biology at the University Medical Center Hamburg-Eppendorf.
A second class of liquid biopsy focuses on tiny membrane-encapsulated packages of RNA and protein called exosomes. Exosomes provide researchers a glimpse of cancer cells’ gene expression patterns, meaning they can reveal differences that are invisible at the DNA level. But, because both normal and cancerous cells release exosomes, the trick, as with ctDNA, is to isolate and characterize those few particles that stem from the tumor itself.
The third counts circulating tumor cells, or CTCs. They are not found in healthy individuals, but neither are they prevalent even in very advanced cases, accounting for perhaps one to 100 per billion blood cells, according to Lim. Researchers can simply count the cells, as CTC abundances tend to scale with prognosis.
But there’s much more that CTCs can do, Pantel says. “You can analyze the DNA, the RNA, and the protein, and you can put the cells in culture, so you can get some information on responsiveness to drugs.” Stefanie Jeffrey, a professor of surgery at Stanford University School of Medicine, has purified CTCs and demonstrated that individual breast cancer CTCs express different genes than the immortalized breast cancer cells typically used in drug development. That, she says, “raises questions” about the way potential drugs are currently evaluated in the early stages of development.
Similarly, Toner and Haber have developed a device called the CTC-iChip to count and enrich CTCs from whole blood. The size of a CD—indeed, the chips are fabricated using high-throughput CD manufacturing technology—these devices take whole blood, filter out the red cells, platelets, and white blood cells, and keep what’s left, including CTCs. The team has used this device to evaluate hundreds of individual CTCs from breast, pancreatic, and prostate tumor patients to identify possible ways to selectively kill those cells.
Elsewhere, Caroline Dive, a researcher at the University of Manchester, has even injected CTCs isolated from patients with small-cell lung cancer into mice. The resulting tumors exhibit the same drug sensitivities as the starting human tumors, providing a platform that could be used to better identify treatment options.
A Range of Uses
According to Lim, liquid biopsies have five potential applications: early disease detection, cancer staging, treatment monitoring, personalized treatment, and post-cancer surveillance. Of those, most agree, the likely near-term applications are personalized treatment and treatment monitoring. The most difficult is early detection.
Among other things, early detection requires testing thousands of early-stage patients and healthy volunteers to demonstrate that the tests are sufficiently sensitive to detect cancer early yet specific enough to avoid false positives. A widely adopted assay that was, say, 90% specific could yield perhaps millions of false positives, Pantel says. “I’m sure that’s fantastic for the lawyers, but not for the patients.”
Still, researchers have begun demonstrating the possibility. In one 2014 study describing a new method for analyzing ctDNA, Diehn, the Stanford radiation oncologist, and his colleague, Ash Alizadeh, an assistant professor of medical oncology also at Stanford, showed that they could detect half of the stage I non-small-cell lung cancer samples it was confronted with, and 100% of tumors stage II and above. That’s despite the fact that ctDNA fragments are only about 170 bases long—a very short amount—and disappear from the blood within about 30 minutes. “There’s constant cell turnover in tumors,” Diehn says. “There’s always some cells dying, and that’s what lets you detect it.”
In another study, Nickolas Papadopoulos, a professor of oncology and pathology at the Johns Hopkins School of Medicine, and his colleagues surveyed the ctDNA content of 185 individuals across 15 different types of advanced cancer. For some tumor types, including bladder, colorectal, and ovarian, they found ctDNA in every patient tested; other tumors, such as glioblastomas, were more difficult to pick up. “It made sense,” Papadopoulos says. “These tumors are beyond the blood-brain barrier…and they do not shed DNA into the circulation.” In later studies, the team demonstrated that some tumors are more easily found in bodily fluids other than blood. Certain head and neck cancers are readily detected in saliva, for example, and some urogenital cancers can be detected in urine. But in their initial survey, Papadopoulos and his colleagues also tested blood plasma for the ability to detect localized (that is, non-metastatic) tumors, identifying disease in between about half and three-fourths of individuals.
Though 50% sensitivity isn’t perfect, it’s better than nothing, Papadopoulos says, especially for cancers of the ovaries and pancreas. “Right now, we get 0% of them because there’s no screening test for these cancers.”
In the meantime, researchers are focusing on personalized therapy. Alizadeh and Diehn, for instance, have tested patients with stage IV metastatic non-small cell lung cancer, a grave diagnosis, who had been taking erlotinib, a drug that targets specific mutations in the EGFR gene. Over time, all patients develop resistance to these drugs, half of them via a new mutation, Diehn says. Diehn and Alizadeh have begun looking for that mutation in the ctDNA of patients whose disease progresses, or returns, as such tumors can be specifically targeted by a new drug, osimertinib. “It’s been shown in a couple of studies that such patients then have a good response rate,” Diehn says, with the median “progression-free survival” doubling from about ten months to 20.
Toward the Clinic
Most scientists working on liquid biopsies agree that the technology itself is mature. What’s needed to make a difference in patients’ lives is clinical evidence of sensitivity, selectivity, and efficacy.
Fortunately, they’re working on it. According to the National Institutes of Health’s clinical trials database, clinicaltrials.gov, over 350 trials are currently studying the use of liquid biopsies in cancer detection, identification, or treatment.
One recent trial, published in April in JAMA Oncology, examined the ability of ctDNA analysis to detect key mutations in two genes associated with treatment decision, response, and resistance in non-small cell lung cancer. The 180-patient prospective trial determined that the method used could detect the majority (64% –86%) of the tested mutations with no false-positive readings in most cases. Results were returned on average within three days, compared to 12 to 27 days for solid-tissue biopsy. The technique is ready for clinical use, the authors concluded.
In an ongoing trial, Pantel and his colleagues are focusing on a breast cancer-associated protein called HER2. Several anticancer therapies specifically target HER2-positive tumors, including trastuzumab and lapatinib. The trial is looking for instances of HER2-expressing CTCs in patients with metastatic breast cancer whose original tumor did not express HER2. About 20% of HER2-negative tumors meet that criterion, Pantel says, but before liquid biopsies became an option, there was really no way to find them. Now, his team is testing “whether the change to HER2-positive CTCs is a good predictor for response to HER2-targeted therapy.” If it is, it could unlock potential treatments for patients.
In another trial, Flaherty, the center director at MGH, and his colleagues are using a series of liquid biopsies in several hundred patients with metastatic melanoma to determine if they could retrospectively predict drug resistance by monitoring for mutations in a particular gene.
In the meantime, diagnostics firms are developing assays of their own. Currently, there is only one FDA-approved liquid biopsy test on the market in the United States. But there also are a growing handful of lab-developed assays for specific genetic mutations available and several more in development.
Early cancer screening is farther out, and while many researchers still express skepticism, the application received a high-profile boost in January when sequencing firm Illumina announced it was launching a spinoff company called Grail. The company, which has already raised some $100 million in funding, will leverage “very deep sequencing” to identify rare ctDNA mutations, and plans to launch a “pan-cancer” screening test by 2019.
Only time will tell, though, whether Grail or any other company is able to fundamentally alter how patients are treated for cancer. But one thing is certain, Flaherty says: Genetic testing, however it is done, only addresses the diagnostics side of the personalized medicine challenge; progress is also required on the drug development side. After all, what good is a test if there’s no way to act on it?
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