Sequencing the fragments of tumor DNA that circulate in blood may give a more accurate picture of a patient’s metastatic cancer than can be obtained from biopsies.

Researchers at the OHSU Knight Cancer Institute recently showed that sequencing cell-free DNA can find the same clinically relevant mutations identified in DNA from tumor tissue, and it can provide additional information about the evolution of a particular patient’s disease and how best to treat it.

That’s significant because drawing blood to obtain cell-free DNA is less invasive and safer for patients than taking a biopsy of tumor tissue. The “liquid biopsy” approach makes it feasible to repeatedly sample tumor DNA over the course of treatment.

“It takes out the risk — and a lot of the cost,” says Paul Spellman, Ph.D., a professor of molecular and medical genetics at OHSU who led the study, published in the open access journal PLOS ONE.

Taking biopsies at multiple metastatic sites often isn’t possible, for instance, when they are located in tissues that are difficult to access. Some lesions are too small to obtain enough tissue. And because tumors often consist of genetically heterogeneous cells, a biopsy needle can miss important mutations that arose outside the sampled area. Cell-free DNA in the blood stream, released from normal and cancerous cells when they die, offers an attractive way around these problems. Sequencing just the protein-coding regions, the exome, makes the process faster and less expensive.

Researchers elsewhere have shown that exome sequencing of cell-free DNA provides information comparable to that obtained from biopsies. But in previous work, tumor DNA made up an unusually large proportion of the cell-free DNA — from 33 to 65 percent — leaving it unclear how practical the approach could be. The OHSU researchers showed that it’s feasible in more typical cases when tumor DNA makes up only a small fraction of the cell-free DNA, less than 8 percent.

Actionable findings

Spellman and colleagues reported results in two patients with metastatic disease, and in both cases cell-free DNA yielded as much or more information about cancer genes as did biopsies. The first patient was 52-year-old woman diagnosed with an inoperable sarcoma of the pulmonary artery who developed metastases in the lungs, liver, and pulmonary arteries. Whole-exome sequencing of cell-free DNA detected 47 of the 48 mutations known from biopsy of the primary tumor, including three significant activating mutations: KRAS G12R, PIK3CA R88Q, and PIK3CA Q546R.

The cell-free DNA also revealed 11 mutations not found in the primary tumor. The researchers were unable to get a sample of the metastasis in this patient, but given the low number of mutations unique to the cell-free DNA, they inferred that there were probably relatively few differences between the metastasis and primary tumor.

The second patient, a 41-year-old woman, had ER positive, HER2 positive breast cancer with metastases in the liver and spine. Sequencing of the primary tumor and liver metastases revealed 48 mutations. Using cell-free DNA in blood, researchers found 38 of these mutations, and another 8 mutations unique to the cell-free DNA that likely arose in hidden metastatic sites (shown in the figure above.)

A PIK3CA mutation showed up only in the primary tumor, not in the liver metastatic site or the cell-free DNA, indicating that the PIK3CA mutation emerged after metastasis or was not present in the tumor cells that seeded the metastasis. The implication is that treatment with a PI3K inhibitor would have been ineffective against any of the distant metastases.

Sequencing revealed an ESR1 mutation in the cell-free DNA and metastatic site, but not in the primary tumor. ESR1 makes cancerous cells resistant to estrogen deprivation therapy, thus, the mutation potentially explains the failure of the aromatase inhibitor the patient received.

Near real-time monitoring

The ability to sequence tumor DNA repeatedly from blood samples would give doctors a near real-time picture of the changing cancer genome during treatment, the researchers say. In theory, identification of emerging mutations would allow patients to start or stop therapies to match changing conditions.

Limitations include uncertainty about how fully cell-free DNA represents the diversity of metastatic disease. “Some metastases may be shedding a lot more tumor DNA than others,” Spellman says. If there are some types of metastases that shed little or no tumor DNA, “we will miss them completely,” he says.

All sequencing approaches face an even more immediate problem: they can reveal any number of altered genes but the information has no value if none of the mutations have been matched to therapies. “Twenty-ish percent of the time, if you sequence the tumor you can find something that’s targetable,” Spellman says. But it’s impossible to know in advance whether undergoing a biopsy procedure will yield useful genetic information that make it worth the risk of bleeding, infection, accidental injury to a nearby organ and other complications. For that reason, a non-invasive method to obtain tumor DNA is urgently needed. With a blood sample biopsy, “you can take the risks down to almost zero,” Spellman says.

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Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease by Timothy M. Butler, Katherine Johnson-Camacho, Myron Peto, Nicholas J. Wang, Tara A. Macey, James E. Korkola, Theresa M. Koppie, Christopher L. Corless, Joe W. Gray, and Paul T. Spellman; PLoS One, August 28, 2015.