Management of metastatic breast cancer requires monitoring of tumor burden to assess response to treatment, and there is a need for biomarkers that can measure tumor burden with high sensitivity and specificity. Assays measuring serum cancer antigen (CA) 15-3 and circulating tumor cells have been reported to have sensitivities of approximately 60% to 70%. As reported in The New England Journal of Medicine, a proof-of-concept study performed by Sarah-Jane Dawson, FRACP, PhD, of the University of Cambridge and the Cancer Research UK Cambridge Institute, and colleagues has shown that circulating tumor DNA is “an informative, inherently specific, and highly sensitive” biomarker of metastatic breast cancer.¹

In this study, the investigators used targeted sequencing to identify somatic genetic alterations in TP53 and PIK3CA, the two genes that are noted to be mutated in up to 50% of all patients with metastatic breast cancer. In a selected number of the remaining individuals, the investigators undertook whole-genome sequencing to identify mutations in other genes or structural variants that were unique to the tumor. They then designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens using digital polymerase chain reaction or targeted deep sequencing. CA 15-3 levels and circulating tumor cell numbers were measured at the same time points.

Computed tomography (CT) imaging of tumors was compared with assays for circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. The 30 women were those from a group of 52 in whom genomic alterations (point mutations [in PIK3CA or TP53] or structural variants) could be identified. Overall, 22 of the 30 had mutations (in PIK3CA or TP53), 3 had mutations and structural variants, and 5 had structural variants only.

Relative Sensitivity

Overall, circulating tumor DNA was detected in 29 (97%) of 30 women and 115 (82%) of 141 plasma samples. The median level of circulating tumor DNA across samples was 150 amplifiable copies/mL (interquartile range, 9–720 copies/mL).

Data comparing CA 15-3 and circulating tumor DNA levels were available for 114 serial time points for 27 patients. CA 15-3 levels were elevated (> 32.4 U/mL) at one or more time points in 78% of women (21/27) and 62% of samples (71/114), whereas circulating tumor DNA was detected in 96% of women (26/27) and 82% of samples (94/114). Of the 43 samples without elevated CA 15-3 levels, 27 (63%) had measurable levels of circulating tumor DNA. Analysis using a modified bootstrapping method showed that the sensitivity of circulating tumor DNA was 85% vs 59% for CA 15-3, with a median difference in sensitivity of 26% (P < .002).

Data comparing circulating tumor cells and circulating tumor DNA levels were available for 126 time points for all 30 women. Circulating tumor cells were detected (≥ 1 cell/7.5 mL) at one or more time points in 87% of women (26/30), with elevated counts (≥ 5 cells/7.5 mL) identified in 60% of women, and in 37% of samples (46/126). By comparison, circulating tumor DNA was detected in 97% of women (29/30) and 84% of samples (106/126). Of the 50 samples in which circulating tumor cells were not detected, circulating tumor DNA was detected in 33 (66%).

On the modified bootstrapping method, sensitivities were 90% for circulating tumor DNA vs 67% for circulating tumor cells, with a median difference in sensitivity of 27% (P < .002). At the median, the number of amplifiable copies of circulating tumor DNA was 133 times the number of circulating tumor cells and exhibited a greater dynamic range.

Tumor Monitoring

The performance of the biomarkers was compared with computed tomography (CT) in 20 patients with measurable disease in whom biomarker data were available at three or more time points over 100 days of follow-up. Circulating tumor DNA was detected in 95% of these women with fluctuations in levels generally correlating with treatment responses observed on CT.

Similar correlations were observed in 10 (50%) of the women who had elevated circulating tumor cell counts (≥ 5 cells/7.5 mL), with the lower counts in the remainder of the group being uninformative with regard to treatment response. Women with high levels of CA 15-3 also had fluctuations corresponding to CT responses, but with a small dynamic range; no consistent serial changes in CA 15-3 levels were observed in the 8 (42%) of 19 patients with levels of 50 U/mL or lower.

Progressive disease was documented by CT in 19 of 20 women during follow-up. Increases in circulating tumor DNA levels reflected progressive disease in 17 (89%) of the 19, with levels increasing by a factor of 505 from the lowest point prior to establishment of progressive disease. By comparison, the numbers of circulating tumor cells increased in 7 (37%) of the 19 women and CA 15-3 levels increased in 9 (50%) of 18.

Circulating tumor DNA levels provided an early measure of treatment failure. In 10 of the 19 patients, circulating tumor DNA levels increased at an average of 5 months (range, 2–9 months) before detection of progressive disease on imaging. In two women, increased levels of circulating tumor DNA did not reflect CT-detected progressive disease.

Prognostic Utility

Proportional hazards analysis showed that increased levels of circulating tumor DNA (P < .001) and increased numbers of circulating tumor cells (P = .03), but not increasing CA 15-3 levels, were significantly associated with poorer overall survival. Increasing circulating tumor DNA levels and increasing numbers of circulating tumor cells were associated with increasing relative hazard values, indicating that absolute levels of each biomarker provided prognostic information.

As stated by the investigators, “In the detection of metastatic breast cancer, circulating tumor DNA shows superior sensitivity to that of other circulating biomarkers and has a greater dynamic range that correlates with changes in tumor burden. Circulating tumor DNA often provides the earliest measure of treatment response, as has been supported by recent analyses of circulating tumor DNA in other solid cancers.”

The investigators concluded:

Our expanding knowledge of the genetic mechanisms underpinning breast cancer now provides a framework to better stratify patients. The analysis of circulating tumor DNA represents a unique opportunity to integrate this knowledge into the clinical arena. Although the acquisition of tumor-tissue specimens will continue to be important, the use of biopsy specimens is limited, since such material may not capture tumor heterogeneity; in addition, repeated biopsy is impractical. Circulating tumor DNA represents a ‘liquid biopsy’ alternative, allowing for sensitive and specific serial sampling to be performed during the course of treatment. ■

Disclosure: Dr. Dawson reported receiving grant support from Australian NHMRC. For a complete list of other study authors’ potential conflicts of interest, visit www.nejm.org.

Reference

1. Dawson S-J, Tsui DWY, Murtaza M, et al: Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 368:1199-1209, 2013.