Cell-free DNA Concentration as a Biomarker of Response and Recurrence in HER2-Negative Breast Cancer Receiving Neoadjuvant Chemotherapy

Abstract

Purpose: We previously demonstrated the clinical significance of circulating tumor DNA (ctDNA) in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC). Here, we compared its predictive and prognostic value with cell-free DNA (cfDNA) concentration measured in the same samples from the same patients.

Experimental design: 145 patients with hormone receptor (HR)-positive/HER2-negative and 138 triple-negative breast cancer (TNBC) with ctDNA data from a previous study were included in the analysis. Associations of serial cfDNA concentration with residual cancer burden (RCB) and distant recurrence-free survival (DRFS) were examined.

Results: In TNBC, we observed a modest negative correlation between cfDNA concentration 3 weeks after treatment initiation and RCB, but none of the other timepoints showed significant correlation. In contrast, ctDNA was significantly positively correlated with RCB at all timepoints (all R > 0.3 and P < 0.05). In the HR-positive/HER2-negative group, cfDNA concentration did not associate with response to NAC, but survival analysis showed that high cfDNA shedders at pretreatment had a significantly worse DRFS than low shedders (hazard ratio, 2.12; P = 0.037). In TNBC, the difference in survival between high versus low cfDNA shedders at all timepoints was not statistically significant. In contrast, as previously reported, ctDNA at all timepoints was significantly correlated with DRFS in both subtypes.

Conclusions: In TNBC, cfDNA concentrations during therapy were not strongly correlated with response or prognosis. In the HR-positive/HER2-negative group, pretreatment cfDNA concentration was prognostic for DRFS. Overall, the predictive and prognostic value of cfDNA concentration was more limited than that of ctDNA.

Figure 1.. Association of cfDNA concentration early during treatment and residual cancer burden (RCB) after neoadjuvant chemotherapy.

CfDNA concentration was measured in the plasma 3 weeks after treatment initiation (T1) in patients with hormone receptor-positive/HER2-negative (HR+HER2−, top panel) and triple-negative breast cancer (TNBC, lower panel); (A, C) Correlation of cfDNA concentration (ng/ mL, log₁₀-transformed) at T1 and RCB index. The blue line and gray shading represent the regression line and the 95% confidence interval, respectively. Correlation coefficient and p values were calculated using Pearson’s correlation test; (C, D) Distribution of cfDNA concentration at T1 by RCB class. RCB was divided into 4 classes: RCB-0, equivalent to pathologic complete response, and −I, −II, −III, representing limited, moderate, and extensive residual cancer, respectively. For each box plot, the center line represents the median value (50th percentile), while the box contains the 25th to 75th percentiles of the data distribution. The whiskers represent the 5th and 95th percentiles, and the dots beyond the upper and lower bounds are considered outliers. Pairwise P values were calculated using the Wilcoxon rank sum test with Bonferroni correction to adjust for multiple comparisons.

Figure 2.. Prognostic significance of pretreatment cfDNA concentration.

Survival analysis in patients with (A, B) hormone receptor-positive/HER2-negative (HR+HER2−) and (C) triple-negative breast cancer (TNBC) stratified into two groups, high vs. low cfDNA-shedders, using the median cfDNA concentration as the cutoff. The survival endpoint was distant recurrence-free survival (DRFS). Hazard ratios (HR) and 95% confidence intervals (CI) were calculated using univariable Cox regression analysis. P values were calculated using the Wald test. The forest plot in B shows HRs and 95% CIs for patients with HR+HER2− breast cancer, estimated from a multivariable Cox regression model that included cfDNA (cfDNA) concentration at pretreatment (T0), adjusted for circulating tumor DNA (ctDNA) status at pretreatment (T0) and residual cancer burden (RCB).

Figure 3.. Clinical significance of cell-free DNA (cfDNA) vs. circulating tumor DNA (ctDNA) in hormone receptor-positive HER2-negative (HR+HER2−) and triple-negative breast cancer (TNBC).

(A) Association of pretreatment (T0) cfDNA (see Supplementary Figure 3) and ctDNA concentration as continuous variables with clinicopathologic variables. The p values were calculated using the Wilcoxon rank-sum test; (B) Association of pretreatment (T0) cfDNA concentration (high vs. low cfDNA shedders using the median as the cutoff, see Table 1) and ctDNA (positive vs. negative) as dichotomous variables with clinicopathologic variables. The p values were calculated using Fisher’s exact test; (C) Correlation of cfDNA and ctDNA concentration (as continuous variables) at different timepoints (T0-T3) vs. residual cancer burden (RCB) index, the continuous measure of residual disease in the breast and regional lymph nodes after NAC. The p values were calculated using Pearson’s correlation test (see Figure 1 and Supplementary Figure 4). The color of the dot represents a negative (red) or positive (blue) correlation; (D) Correlation of cfDNA and ctDNA (as dichotomous variables) at different timepoints (T0-T3) vs. distant recurrence-free survival (DRFS) using Cox regression analysis. The p values were calculated using the Wald test (see Figure 2 and Supplementary Figure 6). The color of the dot represents decreased DRFS (red, hazard ratio>1) or increased DRFS (blue, hazard ratio <1). CfDNA and ctDNA were analyzed in the same plasma sample collected at pretreatment (T0), 3 weeks after treatment initiation (T1), 12 weeks after treatment initiation between paclitaxel-based treatment and anthracycline regimens (T2), and after neoadjuvant chemotherapy before surgery (T3).