Clinical significance and biology of circulating tumor DNA in high-risk early-stage HER2-negative breast cancer receiving neoadjuvant chemotherapy

Abstract

Circulating tumor DNA (ctDNA) analysis may improve early-stage breast cancer treatment via non-invasive tumor burden assessment. To investigate subtype-specific differences in the clinical significance and biology of ctDNA shedding, we perform serial personalized ctDNA analysis in hormone receptor (HR)-positive/HER2-negative breast cancer and triple-negative breast cancer (TNBC) patients receiving neoadjuvant chemotherapy (NAC) in the I-SPY2 trial. ctDNA positivity rates before, during, and after NAC are higher in TNBC than in HR-positive/HER2-negative breast cancer patients. Early clearance of ctDNA 3 weeks after treatment initiation predicts a favorable response to NAC in TNBC only. Whereas ctDNA positivity associates with reduced distant recurrence-free survival in both subtypes. Conversely, ctDNA negativity after NAC correlates with improved outcomes, even in patients with extensive residual cancer. Pretreatment tumor mRNA profiling reveals associations between ctDNA shedding and cell cycle and immune-associated signaling. On the basis of these findings, the I-SPY2 trial will prospectively test ctDNA for utility in redirecting therapy to improve response and prognosis.

Keywords: circulating tumor DNA; gene expression; neoadjuvant chemotherapy; pathologic complete response; receptor subtype; residual cancer burden.

Figure 1.. Predictive value of ctDNA in high-risk early-stage HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC).

A-D. Distribution of ctDNA concentration expressed as mean tumor molecules per ml (MTM/mL) of plasma across time points during NAC in hormone receptor-positive/HER2-negative (HR+HER2−, left panel) and triple-negative breast cancer (TNBC, right panel) patients grouped according to (A, B) pathologic complete response (pCR) and (C, D) residual cancer burden (RCB) class. ctDNA was analyzed in plasma 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 NAC before surgery (T3). For each box plot, the center line represents the median value (50th percentile); 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 outliers; E-J. Patients who were ctDNA-positive at T0 were classified into 3 groups based on ctDNA dynamics from T0 to T2. Distribution of ctDNA dynamic patterns in E. HR+HER2− and F. TNBC patients with complete ctDNA data from T0-T2. Association of ctDNA dynamics with (G, H) pCR and (I, J) RCB. RCB was divided into 4 classes: RCB-0 (no residual disease equivalent to pCR), RCB-I (minimal burden), RCB-II (moderate burden), and RCB-III (extensive burden). Logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval of achieving (G, H) pCR or (I, J) RCB-0/I.

Figure 2.. Prognostic value of ctDNA in high-risk early-stage HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC).

A-D. Survival curves of patients grouped according to combined ctDNA status after NAC (ctDNA-positive or ctDNA-negative at T3) and (A, B) pCR (pCR or no pCR) or (C, D) binarized RCB (RCB-0/I or RCB-II/III) in hormone receptor-positive/HER2-negative subset (HR+HER2−, left panel) and triple-negative breast cancer (TNBC, right panel). RCB was divided into 2 classes: RCB-0 (no residual disease equivalent to pCR) and RCB-I (minimal burden) vs. RCB-II (moderate burden) and RCB-III (extensive burden). Hazard ratios (HR) and 95% confidence intervals (CI) shown were estimated from Cox proportional hazards regression models; E-H. Distribution of ctDNA dynamic patterns in E. HR+HER2− and F. TNBC patients with complete ctDNA data for all time points. Survival curves of G. HR+HER2− and H. TNBC patients grouped according to ctDNA dynamics. Distant recurrence-free survival (DRFS) was the survival endpoint. P-values were calculated using the log-rank test.

Figure 3.. Differentially expressed genes and enriched gene sets associated with ctDNA shedding at pretreatment.

A. GSEA of MSigDB Hallmark gene sets in hormone receptor-positive/HER2-negative subset (HR+HER2−) (top) and TNBC (bottom). Gene sets with significant (BH adjusted p<0.05) and nominally significant (p<0.05) enrichment in ctDNA-positive or ctDNA-negative patients are depicted with p-value significance and normalized enrichment scores (NES). B-C. Gene expression heatmap of leading-edge genes in enriched immune-associated gene sets: B. IL6 JAK STAT3 signaling, inflammatory response, and TNF-alpha signaling via NF-κB in HR+HER2−; and C. Interferon alpha response, and TGFB signaling in TNBC. D. STRING protein-protein interaction (PPI) of genes that are commonly differentially expressed (p<0.05) in both HR+HER− and TNBC with concordant fold change direction across subtypes. The PPI for the 99 genes comprising the main network is shown with community membership, differential expression direction, degree and type of connectivity, and PPI confidence level annotated. Each community’s STRING PPI KEGG functional enrichment is listed.