Abstract PS2-09-18: Real-time liquid biopsy assessment in early-stage breast cancer: methylation based-ctDNA analysis and CTCs detection as potential predictors of response to neoadjuvant therapy

Abstract Background: In early-stage breast cancer (EBC), liquid biopsy may help refine risk stratification and therapeutic decision-making. Historically, the detection of circulating tumor cells (CTCs)>1/7.5 ml has a strong prognostic value for high risk of relapse, but rarely are detected. We explored ctDNA and CTC detection at diagnosis and their association with tumor characteristics and response to neoadjuvant therapy (NAT). Methods: We retrospectively analyzed 40 patients with stage I-III breast cancer who underwent GR and Cellsearch testing at diagnosis and/or during NAT. Guardant Reveal™ (GR) is a tissue-naïve, methylation-based cell-free DNA assay capable of detecting circulating tumor DNA (ctDNA) with high sensitivity, even in low-input samples. CellSearch® detects CTCs via immunomagnetic enrichment of EpCAM-positive cells and cytokeratin-based immunostaining, followed by automated enumeration. Associations between baseline ctDNA and CTC detection and clinical features—including tumor grade, stage, molecular subtype—and response to NAT were assessed. Results: In total, 71 Guardant Reveal™ tests were performed across 40 patients, with a median of 1 test per patient (range 1-7). Thirty patients had a baseline GR before starting NAT. Seventeen patients (43%) underwent more than one GR test during or after NAT, and 15/40 patients (38%) had at least one GR test after surgery. The median time between GR tests was 3.2 months (IQR 2.7-5.0; range 0.3-15.1), while the median interval between surgery and the post-surgery GR test was 3.5 months (IQR 1.9-4.7; range 0.5-8.3). The median age of the cohort was 45 (range 28-77); most patients had invasive ductal carcinoma (88%) and received NAT (85%). Among the 33 patients who underwent testing pre-NAT, ctDNA was detected in 17 (52%). ctDNA detection was significantly associated with tumor grade and molecular subtype, being more frequent in G3 than G1-G2 tumors (72% vs. 27%, p=0.02), andin triple-negative and HR-positive subtypes compared to HER2-enriched (79% vs. 35% vs. 0%, p=0.04). Inflammatory breast cancer and stage III showed a trend toward higher ctDNA tumor fraction (median methylation tumor fraction 0.43% and 0.02%, p=0.04 and 0.05, respectively). Lower ctDNA levels at diagnosis were observed in patients achieving pCR (pCR vs. no pCR, median methylation tumor fraction 0.03% vs. 0.09%), although the association is not statistically significant (p=0.26). Conclusion: CTC testing with CellSearch® was performed in 29 patients, for a total of 56 tests. 15 patients (52%) underwent more than one CTC test, enabling longitudinal assessment. Among CTC-tested patients, only one patient was positive (11 CTC/7.5mL), converting to negative after 2 months of NAT. No additional CTC positivity was observed during follow-up. At the time of analysis, no recurrences had been observed (median follow-up: 13.6 months; range 6.6-18.2 from diagnosis to last contact) and four patients are still receiving NAT. Conclusion: In EBC, ctDNA detection using a methylation-based assay identified patients with aggressive features and potential lower likelihood of achieving pCR. The use of methylation-based ctDNA testing may hold promise as a tool to guide escalation or de-escalation strategies, supporting a more personalized approach to treatment when combined with other biomarkers such as CTCs. Further validation in larger cohorts is needed. Updated data will be presented at the conference. Citation Format: C. Gianni, I. Azzali, L. Pontolillo, B. Pastò, E. Nicolo', M. Serafini, N. Bayou, M. Velimirovic, A. J. Medford, M. D. Lipsyc-Sharf, A. Davis, C. Reduzzi, A. Musolino, A. Bardia, M. Cristofanilli. Real-time liquid biopsy assessment in early-stage breast cancer: methylation based-ctDNA analysis and CTCs detection as potential predictors of response to neoadjuvant therapy [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-09-18.