Liquid biopsy is a test done on a sample of blood to look for cancer cells from a tumor that are circulating in the blood or for pieces of DNA from tumor cells that are in the blood. A liquid biopsy may be used to help find cancer at an early stage. It may also be used to help plan treatment or to find out how well treatment is working or if cancer has come back. Being able to take multiple samples of blood over time may also help doctors understand what kind of molecular changes are taking place in a tumor.

Circulating tumor DNA (ctDNA) is found in the bloodstream and refers to DNA that comes from cancerous cells and tumors. Most DNA is inside a cell’s nucleus. As a tumor grows, cells die and are replaced by new ones. The dead cells get broken down and their contents, including DNA, are released into the bloodstream. ctDNA are small pieces of DNA, usually comprising fewer than 200 building blocks (nucleotides) in length.

The quantity of ctDNA varies among individuals and depends on the type of tumor, its location, and for cancerous tumors, the cancer stage.

Detection of ctDNA can be helpful in the following cases:

• Detecting and diagnosing a tumor. Because tumor DNA has acquired multiple genetic mutations, leading to tumor development, ctDNA is not an exact match to the individual’s DNA. Finding DNA with genetic differences aids in tumor detection. Diagnosing the type of tumor using ctDNA can reduce the need for getting a sample of the tumor tissue (tumor biopsy), which can be challenging when a tumor is difficult to access, such as a tumor in the brain or lung.

• Guiding tumor-specific treatment. Analyzing the genome of tumor cells using ctDNA can help doctors determine which treatment will be most effective. Currently, however, approval from the U.S. Food and Drug Administration for ctDNA testing to personalize cancer treatment is limited.

• Monitoring treatment. A decrease in the quantity of ctDNA suggests the tumor is shrinking and treatment is successful.

• Monitoring periods with no symptoms (remission of cancer). A lack of ctDNA in the bloodstream indicates that the cancer has not returned.

When a patient has a suspicious lump or symptoms, one of the first things a doctor may do is perform a tissue biopsy—a procedure to collect cells for closer examination.

Examining the appearance of the cells under the microscope can determine if cancer is present, show what type of cancer it is, and give clues about the patient's prognosis. In addition, molecular analysis of a tissue biopsy sample can also reveal information that may help guide a personalized treatment strategy.

Although they are important for patient care, tissue biopsies—which may involve a large needle, an endoscope, or open surgery—can be invasive, risky, costly, and painful. And some patients may not be able to have a tissue biopsy due to the inaccessibility of their tumors or because they have other health conditions that prevent them from undergoing the procedure.

Because these factors make it difficult to perform repeated biopsies on a patient, these tests can be an impractical method to track tumors as they develop and change over time. Nevertheless, they remain the gold standard for detecting and obtaining information about cancer.

But researchers have been exploring a new approach that could potentially complement or, in some cases, serve as an alternative to tissue biopsies. The approach, often called a liquid biopsy, relies on analyzing bits of tumor material—molecules as well as whole cells—that are found in bodily fluids such as blood or urine.

Although there is a widespread belief that liquid biopsies could eventually have a significant impact on patient care, most researchers in the field agree that the science around the approach is still evolving and important questions remain unanswered.

"I think the major stumbling block for moving these liquid biopsy tests forward is there is not enough clinical verification and validation to know and feel comfortable that what we’re detecting with them is clinically meaningful," said Lynn Sorbara, Ph.D., of NCI's Division of Cancer Prevention.

Different Tests for Different Tumor Molecules

More than 100 years ago, scientists discovered that tumors shed molecules and cells into bodily fluids. Much more recently, researchers have shown that analyzing these molecules and cells can reveal some of the same information that tissue biopsies provide.

Liquid biopsy research has recently expanded, generating an entirely new field of study. Both academic and industry researchers from diverse areas of expertise are working on many fronts to develop, refine, and establish clinical uses for liquid biopsy tests.

Different liquid biopsy tests analyze different kinds of tumor material, such as DNA, RNA, proteins, tiny vesicles called exosomes, and whole cells. The tests detect these molecules or cells in various bodily fluids, including blood, urine, cerebrospinal fluid, or saliva. These body fluids are usually readily accessible, and in most cases the procedure for collecting a sample is less invasive and more easily repeatable than a tissue biopsy.

This feature gives liquid biopsies the potential to be used for several important applications for which tissue biopsies are not well suited, explained Miguel Ossandon, M.S., program manager for the Cancer Diagnosis Program in NCI's Division of Cancer Treatment and Diagnosis.

For example, liquid biopsies could be used to monitor cancer development, track a patient's response to treatment, or as a "surveillance" method for people who have completed treatment but are at high risk of their disease returning, he said.

"The variety of technologies emerging to enable more precise and robust analysis of circulating molecules, cells, and everything in-between, certainly suggests that the exciting clinical potential for liquid biopsy approaches is more a question of 'when' rather than 'if'," said Tony Dickherber, Ph.D., director of NCI's Innovative Molecular Analysis Technologies Program.

There has been a recent surge of research related to liquid biopsy tests that analyze tumor DNA in blood, called circulating tumor DNA (ctDNA), and several ctDNA-based liquid biopsy tests are in clinical development.

Using Tumor DNA to Detect Cancer Early

One potential application of ctDNA-based liquid biopsies is for detecting cancer at an early stage, when treatment may be most successful. In several studies, for example, liquid biopsy tests detected ctDNA in blood samples collected from patients months before they were diagnosed with cancer by traditional methods, such as imaging tests.

But, in these studies, the tests sometimes produced false-positive test results—that is, they detected cancerous DNA when no cancer actually developed.

Another concern is that these tests will detect early-stage tumors that will not grow much or will grow so slowly that they would never actually harm the patient.

Treating these slow-growing tumors could actually do more harm than good, and "the risk of overtreatment is a major concern with early cancer detection," Dr. Sorbara noted. "The idea of diagnosing somebody using liquid biopsy alone has not been validated yet. We're still at the early stages and have a long way to go," she continued.

Prospective cohort studies are needed to truly determine if the presence of ctDNA in a patient’s blood can be used as an accurate marker for early-stage cancer, she added. For example, studies are needed to determine if the detection of ctDNA warrants treatment and if that treatment improves patient outcomes.

NCI is supporting an initiative to advance the development and validation of liquid biopsytechnologies that can detect early-stage cancers, distinguish cancer from benign conditions, and identify fast- and slow-growing cancers. A major aim of the initiative is to create a public–private partnership that brings engineering and clinical experts together to accomplish these goals.

Looking forward, Dr. Sorbara envisions that liquid biopsy tests may be used to screen for early-stage cancer in high-risk individuals, such as those with hereditary cancer syndromes.

Or, she continued, they could be used in tandem with other tests, such as an MRI. For example, a liquid biopsy test could be used as a routine prescreening method in healthy individuals to identify those who may have early-stage cancer and are candidates for other (possibly more costly or invasive) screening tests.

Tumor DNA May Aid Precision Cancer Treatment

There is also hope that ctDNA-based liquid biopsies may guide precision medicine treatment by identifying unique molecular characteristics of an individual's cancer. In several research studies, liquid biopsies have pinpointed ctDNA mutations that could potentially be used to determine the optimal treatment.

For example, researchers at UC San Diego Moores Cancer Center analyzed blood samples from 168 patients with different types of cancer, including brain, lung, and breast cancer. For 58% of the participants, the researchers identified at least one cancer-related ctDNA mutation. For most of these patients, a Food and Drug Administration (FDA)–approved drug was available to treat cancers with that particular mutation.

Other studies have demonstrated the feasibility of using ctDNA-based liquid biopsies on a large scale to identify DNA mutations in patients' cancers. For instance, investigators used Guardant360—a commercially available test that analyzes 70 cancer-related genes in a blood sample—to identify mutations in the ctDNA of more than 15,000 patients. The investigators found that, for most patients, the genetic mutations identified by the liquid biopsy test were consistent with those identified by a tissue biopsy test.

In 2016, FDA approved a liquid biopsy test, called the cobas® EGFR Mutation Test for the detection of EGFR gene mutations in ctDNA of patients with lung cancer. The purpose of the test is to identify patients who may be candidates for treatment with erlotinib (Tarceva^®) and osimeritinib (Tagrisso^®)—targeted therapies that attack cancer cells with EGFR mutations. Because the test may produce a false-negative test result, FDA recommends a tissue biopsy if the liquid biopsy is negative (meaning it does not detect an EGFR mutation).

Many other liquid biopsy tests are commercially available but have not been rigorously tested by scientists. Clinicians and researchers are still determining the limitations of these tests and, more importantly, whether they provide clinical benefit to patients. For example, it is unknown whether using a liquid biopsy test to help select treatment improves patient outcomes.

Monitoring Treatment Response with Tumor DNA

Because they are noninvasive and easily repeated, ctDNA-based liquid biopsies may be useful for monitoring patients' responses to therapy both during treatment and after it is completed. Clinicians are hopeful that tracking a patient’s response to treatment may allow adjustments to be made in real time. In other words, the treatment could be stopped or adjusted if the test indicates it is not working.

Imaging techniques such as CT scans are currently used to track treatment response for patients with certain cancer types, but they are not sensitive enough to detect small changes in tumor size and they tend to be costly, explained Mark Roschewski, M.D., of NCI's Center for Cancer Research.

As a potential alternative, Dr. Roschewski and his colleagues tested the ability of a liquid biopsy test to track treatment responses in patients with lymphoma. They showed that changes in ctDNA correlated with positive responses to chemotherapy. Furthermore, they were able to use ctDNA patterns to detect when some patients’ disease was coming back—months before it was possible to do so via CT scan.

"In our study, the liquid biopsy test was much more sensitive than imaging techniques," said Dr. Roschewski.

Likewise, other NCI researchers correlated changes in ctDNA levels with patients' responses to immunotherapy treatment. They found that they could detect these changes within 2 weeks of the start of treatment. Having an early indicator of the treatment's efficacy could be very helpful because only a small proportion of patients typically respond to immunotherapy treatment, they explained.

"Liquid biopsy tests have the added advantage of providing molecular information about the cancer, which can change during and after treatment," said Brian Sorg, Ph.D., also of NCI's Division of Cancer Treatment and Diagnosis. This additional information could potentially help doctors track the development of drug resistance and make more personalized treatment decisions.

For example, although most patients with lung cancer initially respond to treatment with a class of drugs called tyrosine kinase inhibitors, the majority develop drug resistance within 1 or 2 years of starting treatment.

In one study, a team of researchers analyzed mutations in ctDNA from patients with lung cancer that had become resistant to certain tyrosine kinase inhibitors. They detected a genetic mutation causing the drug resistance in ctDNA from 80% of participants.

A separate study will be needed to determine if the liquid biopsy test can identify patients whose tumors have this mutation and who are most likely to benefit from a different treatment, the researchers noted.

According to an expert review released by the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), most liquid biopsy tests are not yet ready for routine use in clinical cancer care. Based on an analysis of 77 liquid biopsy studies, the organizations concluded that most liquid biopsy tests designed to track treatment responses need more evidence to confirm that the tests correctly identify patients for whom a particular treatment is likely to be effective. The evidence on the validity of these tests is "still emerging," they wrote.

Limitations of ctDNA-based Liquid Biopsies

While there are many potential applications for ctDNA-based liquid biopsies, there are also several limitations.

Most cancer types lack well-established biomarkers (such as a specific DNA mutation) that allow scientists to identify and track the disease via ctDNA. For example, a biomarker commonly used to track advanced pancreatic cancer is considered unreliable for early detection of the disease.

"While the technology for detecting ctDNA in body fluids has improved dramatically, the knowledge required to identify appropriate biomarkers for many cancer types has not," said Dr. Dickherber.

And DNA mutations vary even among patients with the same cancer type, so although a particular mutation may be common for one type of cancer, many patients with that cancer type may not have it. This adds a layer of complexity to the challenge of identifying ctDNA biomarkers for every cancer type and stage.

One possible solution could involve combining tissue and liquid biopsies, said Ossandon. First, a tissue biopsy could be used to identify unique biomarkers for an individual’s tumor, he explained, and then liquid biopsy tests could be used to track those biomarkers.

Another limitation is that ctDNA in the blood may not be truly representative of DNA in the actual tumor, and, therefore, may not be the best source of information for guiding clinical decisions. Tumors are heterogeneous—meaning DNA mutations vary between cancer cells in a single tumor—and it is not known whether ctDNA is released from the whole tumor or only certain parts of it, explained Dr. Sorg.

It is also unknown whether the mutations found in ctDNA are "driver" mutations—those that play an important role in the cancer's biology—said Dr. Sorbara. They may instead be "passenger" mutations, that is, changes that accompany the development of cancer but do not control its growth.

The ASCO and CAP review also found that the results of most liquid biopsy tests don't completely match results from tissue biopsies, calling into question the accuracy of these new tests. This discordance could be the result of biological differences (between blood and tissue samples, for example) or limitations of the tests, the experts noted.

"Possibly the biggest unanswered question is whether liquid biopsy tests can improve patient survival," Dr. Roschewski said. Meaning, does using liquid biopsy tests to detect early-stage cancer, select treatment, or track disease progression ultimately extend patient survival or improve quality of life?

Many researchers, including the ASCO and CAP experts, agree that studies that prospectively analyze the effect of liquid biopsy tests on clinical outcome are needed.

For example, Stanford University and NCI are leading a clinical trial to evaluate the clinical response and overall survival of patients who receive targeted therapy based on molecular information identified via a tissue or liquid biopsy test. As of October 2017, the trial is currently recruiting adults with metastatic solid tumors.

Women who had cancer cells detected in their blood 5 years after a breast cancer diagnosis were 13 times more likely to have their cancer return than women who did not, results from an ongoing study have shown.

Most women who are diagnosed with hormone receptor–positive breast cancer that has not spread will not have a recurrence. Among those who do have a recurrence, more than half have a late recurrence, meaning their disease will return 5 or more years after diagnosis.

Doctors do not currently have a reliable method to predict who is likely to have a late recurrence and is a candidate for therapies that might prevent or delay it. According to the study findings, a blood test—a type of liquid biopsy—may help classify patients according to their recurrence risk.

“This [finding] is not something that should change practice now,” said lead investigator Kathy Miller, M.D., of Indiana University Melvin and Bren Simon Cancer Center. “But it gives us the potential for future clinical trials that identify patients at higher risk [of late recurrence] and study whether having that information could change treatment and improve outcomes,” she added.

Though there are limitations, “this is a really well-conducted study,” said Lyndsay Harris, M.D., of NCI’s Division of Cancer Treatment and Diagnosis, who has conducted similar research but was not involved in the study. “It’s part of a clinical trial and therefore was carefully controlled.”

The finding that detection of tumor cells in blood is associated with late recurrence of breast cancer “is a really important observation that had not been clearly shown in the setting of a large clinical trial,” she added.

The study, funded in part by NCI, was reported in JAMA Oncology on July 26.

Detecting Breast Cancer Cells in Blood

Tumors are solid masses, but they aren’t static. In addition to growing, spreading, and changing the environment around them, tumors also shed cells into nearby blood vessels. Studies have suggested that measuring and analyzing these so-called circulating tumor cells may reveal important information about the tumor that could benefit the patient.

Liquid biopsies are being widely studied as a way to improve early detection of cancer, monitor disease progression, and help guide treatment decisions.

Dr. Miller and her colleagues wanted to determine whether a liquid biopsy test for circulating tumor cells was associated with late recurrence in women with breast cancer.

More than 700 women who were participating in a large NCI-funded clinical trial of adjuvant therapy for breast cancer also enrolled in this new study. At the time that they enrolled—around 5 years after their original breast cancer diagnosis—the women did not have any evidence of a recurrence.

The researchers used a liquid biopsy test to identify and count tumor cells in a sample of blood collected from participants at the time of enrollment. The test is cleared by the Food and Drug Administration to assess disease progression in patients with metastatic breast, prostate, and colon cancer. However, it has not been proven that using the test to inform treatment decisions improves patient outcomes, Dr. Harris stressed.

Of the 547 women whose samples could be analyzed, 26 had at least one detectable circulating tumor cell. The researchers did not observe any major differences in characteristics such as tumor size or age at diagnosis between the women who had detectable circulating tumor cells and those who didn’t. The proportion of women with hormone receptor–positive (HR+) and hormone receptor–negative (HR–) breast cancer who had detectable circulating tumor cells was similar.

At a median follow up of 2.6 years after the test was performed, cancer had returned in 24 women in the study, including 7 women who had detectable circulating tumor cells and 17 who did not. For all women in the study, the detection of circulating tumor cells was associated with a 12.7-fold higher risk of recurrence.

Late recurrences are typically more common among women with HR+ than HR– breast cancer. Dr. Miller and her colleagues observed the same pattern: cancer returned in 6.5% of women with HR+ breast cancer and 0.5% of those with HR– breast cancer.

Of the 23 women with HR+ breast cancer who had a late recurrence, 7 had detectable circulating tumor cells. For women with HR+ breast cancer, the detection of circulating tumor cells was associated with a 13.1-fold higher risk of recurrence.

Among women with HR+ breast cancer who had detectable circulating tumor cells, those who had a recurrence had higher numbers of circulating tumor cells than those who did not have a recurrence.

None of the eight women with HR– disease who had detectable circulating tumor cells had a recurrence. It’s not clear why circulating tumor cells are associated with recurrence only for women with HR+ disease, Dr. Miller said.

Though there’s no evidence yet, one idea is that the immune system may be better at attacking and eliminating HR– tumors before they have a chance to grow back, she added.

Predicting At-Risk Patients

The research team acknowledged that the study population is small and that they need to follow the participants for a longer time. They are continuing to follow these participants.

“More patients may have a recurrence with further follow up,” Dr. Miller said.

The researchers also plan to determine if testing patients more than once is a more accurate way to predict recurrence. Circulating tumor cells might be detected later in women who had a recurrence but did not test positive at the 5-year mark, said Dr. Miller.

In addition, the team will evaluate other tumor biomarkers—such as certain proteins in the blood—that might be associated with the risk of recurrence “to see if we can do an even better job of segregating patients based on their risk,” Dr. Miller explained.

Dr. Harris noted that the question of how the detection of circulating tumor cells might change patient treatment and, ultimately, outcomes, still needs to be addressed.

Women with HR+ breast cancer are sometimes treated with hormone therapy for more than 5 years in an effort to prevent or delay a late recurrence. But it isn’t clear if all patients with HR+ breast cancer need such treatment, which comes with the risk of serious side effects, for that length of time.

Having a test that could predict which HR+ breast cancer patients are most at risk for a late recurrence and might be good candidates for extended therapy, or another treatment approach, would be helpful, Dr. Harris added.

Dr. Miller and her colleagues are beginning to talk about just such a study, using the liquid biopsy test to determine patient treatment.