Longitudinal evaluation of circulating tumor DNA in patients undergoing neoadjuvant therapy for early breast cancer using a tumor-informed assay

Abstract

Circulating tumor DNA (ctDNA) is an emerging biomarker for the treatment of early breast cancer (EBC). We sought to evaluate a highly sensitive tumor-informed ctDNA assay in a real-world cohort of patients receiving neoadjuvant therapy (NAT) to assess clinical validity and explore prognostic outcomes. ctDNA is detected in 77.2% (88/114) of participants at baseline, with 18/88 (20.5%) having a baseline estimated variant allele frequency (eVAF) of <0.01%. Persistent detection of ctDNA, measured midway through NAT (mid-NAT), is associated with disease recurrence in all participants, reaching statistical significance in those with HER2-negative disease. Stratified analyses demonstrate that ctDNA detected mid-NAT enhances the prognostic accuracy of the residual cancer burden (RCB) score for disease recurrence. Postoperative or follow-up detection of ctDNA demonstrates a 100% positive predictive value for disease recurrence, with a median lead time of 374 days (range: 13-1010 days). These data suggest that assays with high analytical sensitivity may improve baseline ctDNA detection in patients with EBC. The ability to replicate the prognostic association of ctDNA dynamics in a real-world cohort supports further investigation. Prospective trials incorporating ctDNA testing are warranted to assess and develop the clinical utility of ctDNA-guided treatment strategies.

Fig. 1. Representative CONSORT diagram of participants identified for the cohort.

ER+: estrogen receptor positive/HER2-negative, TNBC triple negative breast cancer, HER2+: human epidermal growth factor receptor positive.

Fig. 2. ctDNA quantification and panel characteristics with genomic background.

A Distribution of eVAF across baseline timepoints evaluated (n = 114 baseline samples). B Number of variants included in personalized ctDNA panels, by receptor subtype (One-way ANOVA, p = 0.0003; ER+ n = 40, TNBC n = 29, HER2+ n = 45). C Number of variants per personalized panel in those with and without ctDNA detected at baseline, by receptor subtype (t-test, p=ns; ER+/ctDNA+ n = 28, ER+/ctDNA- n = 12, TNBC/ctDNA+ n = 26, TNBC/ctDNA- n = 3, HER2+/ctDNA+ n = 34, HER2+/ctDNA- n = 11). D Baseline eVAF distribution in all participants and by receptor subtype (One-way ANOVA, p = 0.68; ER+ n = 40, TNBC n = 29, HER2+ n = 45). eVAF in baseline samples for participants by: E clinical T-size (One-way ANOVA, p = 0.082; T1 n = 13, T2 n = 61, T3 n = 32, T4 n = 8), F clinical stage (One-way ANOVA, p = 0.19; IA n = 6, IIA n = 27, IIB n = 50, IIIA n = 20, IIIB n = 7, IIIC n = 4), G clinical N-stage (One-way ANOVA, p = 0.048; N0 n = 42, N1 n = 60, N2 n = 7, N3 n = 4) H Nottingham clinical grade as assessed on diagnostic biopsies (One-way ANOVA, p = 0.039; Grade 1 or 2 n = 32, Grade 2 to 3 n = 22, Grade 3 n = 60) I Recurrence status (t-test, p = 0.024; Recurrence n = 19, No Recurrence n = 95). J Representative Oncoplot of common mutations in breast cancer-related genes, clinical receptor subtype, mutation classification, TMB (mut/Mb), and baseline detection of ctDNA. Box plots demonstrate median, minimum, and maximum values. All values are illustrated individually where horizontal lines represent the median. All p-values are 2-sided, with p < 0.05 indicating statistical significance. ER+: estrogen receptor positive/HER2-negative, TNBC triple negative breast cancer, HER2+ human epidermal growth factor receptor positive, ctDNA+ ctDNA detected, ctDNA- ctDNA not detected, NS not significant, T clinical tumor size, N clinical nodal status.

Fig. 3. Prognostic assessment of ctDNA detection at baseline and mid-NAT.

A Baseline ctDNA detection and association with RFI (HR: 2.89, 95% CI: 1.003–8.31; Log-rank (Mantel–Cox) p = 0.049). B Mid-NAT ctDNA detection and association with RFI in all participants (HR: 3.59, 95% CI: 0.99–12.87; Log-rank (Mantel–Cox) p = 0.050). C Mid-NAT ctDNA detection and association with RFI in participants with HER2-negative disease (HR: 14.43, 95% CI: 3.00–69.53; Log-rank (Mantel-Cox) p = 0.0009). HR hazard ratio, 95% CI: 95% confidence interval, NR not reached.

Fig. 4. Prognostic assessment of ctDNA detection mid-NAT stratified by RCB.

A Mid-NAT ctDNA clearance and RFI in all participants stratified by RCB-score (Log-rank (Mantel–Cox) p = 0.011). Note: curves for RCB-0 Detected and Not Detected are superimposed. B Mid-NAT ctDNA clearance and RFI in participants with HER2-negative EBC stratified by RCB-score (Log-rank (Mantel–Cox) p = 0.0016). Note: no participants with RCB-0 disease had ctDNA detected and are thus not represented. NR not reached, RCB residual cancer burden, RFI recurrence-free interval.

Fig. 5. ctDNA trajectories and clinical events.

Participants’ clinical timeline and timeline of plasma collection for ctDNA analysis for A ER+ B TNBC and C HER2 + EBC. Participants who received adjuvant HER2-targeted therapy who were not classified as HER2+ at baseline had ASCO/CAP-defined HER2+ disease on surgical resection and received one year of trastuzumab as standard of care. RCB residual cancer burden, cN clinical nodal status at baseline, cT clinical tumor size at baseline.

Fig. 6. Landmark detection of ctDNA in postoperative or follow-up timepoints.

A The association between RFI and the detection of ctDNA at postoperative and/or follow-up timepoints (Log-rank (Mantel–Cox) p < 0.0001). NR not reached.

Fig. 7. Dynamic changes in eVAF time.

Representative time course plots illustrating changes in eVAF over time with respect to initiation or changes in therapy for representative patients of interest: A a participant with a diagnosis of ER-low EBC on biopsy, TNBC on resection (B) a participant with ER + EBC with a routine endocrine therapy switch (C) a participant with ER + EBC on adjuvant AI and (D) a participant with TNBC and rapid ctDNA clearance on NAT followed by pre-operative detection. C cycle, FU follow-up pre-op: preoperative time point, post-op postoperative time point, ET endocrine therapy.