ctDNA Biomarker for Responses, Outcomes Observed for High-Risk Patients With Early Breast Cancer

Following neoadjuvant chemotherapy with or without pembrolizumab in patients with high-risk early-stage breast cancer, the presence of ctDNA was a biomarker for response and distant recurrence-free survival.

Following neoadjuvant chemotherapy with or without pembrolizumab (Keytruda) in patients with high-risk early-stage breast cancer, the presence of circulating tumor DNA (ctDNA) was a biomarker for response and distant recurrence-free survival (DRFS), according to findings from a biomarker analysis of the I-SPY 2 trial presented during the 2020 San Antonio Breast Cancer Symposium.1

Investigators led by Mark Jesus M. Magbanua, PhD, a postdoctoral researcher at the University of California San Francisco, determined that early clearance of ctDNA during neoadjuvant chemotherapy treatment was significantly associated with increased likelihood of achieving pathological complete response (pCR). Furthermore, ctDNA clearance appeared to be an early surrogate marker for therapy response assessment, and residual ctDNA after neoadjuvant treatment was a significant predictor for metastatic recurrence and death.

“What this study shows is that personalized monitoring of ctDNA during neoadjuvant chemotherapy is feasible and provides information that can be combined with imaging and pathology and may help to optimize decision-making for de-escalation or escalation of therapy,” Christos Sotiriou, MD, PhD, head of the Institut Bordet J. C. Heuson Breast Cancer Translational Research Laboratory, Université Libre de Bruxelles, in Belgium.

In results from I-SPY 2 (NCT01042379) published in May, investigators concluded that adding pembrolizumab to neoadjuvant chemotherapy more than doubled the estimated pCR rates for both hormone receptor–positive/HER2 (ERBB2)-negative and triple-negative breast cancer.2Magbanua and colleagues hypothesized that ctDNA may be a predictive biomarker for response and survival.

Investigators used a personalized Signatera assay to detect patient-specific variants from whole exome sequencing of pretreatment tumor specimens that provides a ctDNA test customized for the patient’s clonal mutational signature.1,3 Overall, 511 serial plasma samples collected from 138 patients with high-risk hormone receptor–positive/HER2-negative (n = 77) or triple-negative (n = 61) stage II/III breast cancer. Patients received pembrolizumab plus paclitaxel followed by chemotherapy with anthracyclines (n = 42) or standard neoadjuvant chemotherapy only (n = 96).

Plasma was collected at pretreatment (T0), 3 weeks after treatment initiation (T1), prior to administration of anthracyclines (T2), and prior to surgery (T3). ctDNA positivity was defined as detection of a minimum of 2 patient-specific tumor mutation fragments in cell-free DNA.

ctDNA results were available at T0 and T1 for 96% (132/138) of patients. Results were available for 86% (118/136) at T0, T1, and T2.

Compared with patients who were ctDNA-negative at T1, those who were ctDNA-positive were at significantly increased risk for metastatic recurrence and death (HR, 4.35; 95% CI, 1.27-14.69; P = .0097). All patients who achieved pCR were ctDNA-negative at T3 (n = 34). Among the 81 patients who did not reach pCR, DRFS was significantly better among the 72 who were ctDNA-negative compared with those who were ctDNA-positive (HR, 0.13; 95% CI, 0.05-0.37).

Patients who were ctDNA-positive at baseline and their cleared ctDNA levels by T1 had the highest pCR rates at 54%. pCR was 38% for patients were negative for ctDNA at T0 and T1; those who did not clear ctDNA by T1 had the lowest pCR rate of 14%).

Among patients who were ctDNA-positive at baseline, pCR was significantly associated with clearance at T1 (OR = 1.92). Investigators found that the association held after adjusting for hormone receptor status and treatment (likelihood ratio [LR], P <.001) and treatment arm (pembrolizumab: LR, P = .03; control: LR, P = .01).

In patients who were ctDNA-negative at T0 to T2, the pCR rate was 41%. pCR rates were highest at 50% in those with early clearance, followed by those with late clearance (19%) and those without clearance (7%).

The correlation between ctDNA and clinical outcomes also was demonstrated in data that I-SPY 2 investigators published in November from an analysis of 291 plasma samples collected from 84 patients with high-risk early breast cancer treated with standard neoadjuvant chemotherapy alone or combined with the AKT inhibitor MK-2206.4

Investigators collected blood samples pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), and prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole exome sequencing of pretreatment tumor) in cell-free DNA by ultra-deep sequencing.4

Sixty-one (73%) patients were ctDNA-positive at baseline. ctDNA decreased at each successive collection point (T1, 35%; T2, 14%; T3, 9%). Patients who remained ctDNA-positive at T1 were significantly more likely to have residual disease after treatment compared with those who cleared ctDNA (OR, 4.33; P = .012). All patients who achieved pCR (n = 17) were ctDNA-negative and reached a favorable DRFS.

Of the 43 patients who did not achieve pCR, 86% were ctDNA-negative at T3. DRFS was 93%, nearly identical to those who achieved pCR (HR, 1.4; 95% CI, 0.15-13.5).However, those who did not achieve pCR, ctDNA positivity was associated with a significantly worse DRFS (HR, 10.4; 95% CI, 2.3–46). Sixty-seven percent of those who were ctDNA positive at T3 experienced metastatic recurrence.4

“This study shows that personalized ctDNA monitoring is a powerful new tool that can help evaluate novel therapies faster, in conjunction with other biomarkers like pCR and MRI imaging response,” said coauthors Laura J. Esserman, MD, MBA, and Laura J. van ‘t Veer, PhD. Both are professors at the University of California San Francisco and Giants of Cancer Care® award winners. “This technology may also help guide clinical decisions in the neoadjuvant setting, including the type and timing of surgery and whether to switch treatment modalities,” they said.3


  1. Magbanua MJM, Wolf D, Renner D, et al. Personalized circulating tumor DNA (ctDNA) as predictive biomarker in high-risk early stage breast cancer (EBC) treated with neoadjuvant chemotherapy (NAC) with or without pembrolizumab (P). Presented at: 2020 San Antonio Breast Cancer Symposium; December 8-11, 2020. Abstract PD9-02.
  2. Nanda R, Liu MC, Yau C, et al. Effect of pembrolizumab plus neoadjuvant chemotherapy on pathologic complete response in women with early-stage breast cancer: an analysis of the ongoing phase 2 adaptively randomized I-SPY2 trial. JAMA Oncol. 2020;6(5):676-684. doi:10.1001/jamaoncol.2019.6650
  3. New publication from I-SPY 2 trial highlights Signatera's ability to predict treatment outcomes in neoadjuvant breast cancer patients. News release. Natera, Inc; November 30, 2020. Accessed December 11, 2020. https://prn.to/3lZoUOk
  4. Magbanua MJM, Swigart LB, Wu HT, et al. Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival [published online November 20, 2020]. Ann Oncol. doi:10.1016/j.annonc.2020.11.007