Estrogen receptor (ER) expression on circulating tumor cells (CTCs) and cell free DNA (cfDNA) mutational landscape in the PACE randomized phase II study

Background: The PACE (NCT03147287) randomized phase II trial investigates CDK4/6 inhibition beyond progression in combination with endocrine treatment, with or without PD-L1 inhibition, in hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) (Mayer et al 2024). We previously reported that cfDNA alterations and CTC number correlated with survival and treatment response (Jeselsohn et al 2024; Gerratana et al ASCO 2023). Here we investigated ER expression on CTCs in relation to the cfDNA mutational landscape. Methods: Samples were collected at baseline. CTC enumeration and ER protein expression on CTCs (by immunofluorescence) was evaluated with the CellSearch and the ACCEPT software. Samples were classified as CTC^high or CTC^low (cutoff $5 CTC/sample). CTC^high samples were defined ER+ if.15% CTCs/sample expressed ER to ensure good inter-group stratification. Concurrently, cfDNA was analyzed with the Guardant360 assay. Only pathogenic single nucleotide (snv) and copy number variations (cnv) with $3% prevalence were included and categorized into oncogenic pathways (Sanchez-Vega et al 2018). Differences in distribution across CTC groups were tested through Chi-squared and Fisher’s test. Results: From 220 enrolled patients, 167 were evaluable for ER on CTCs. Of these, 91 were CTC^low, 30 were CTC^high/ER-, and 46 CTC^high/ER+. ESR1 mutations were more common in CTC^high/ER+ samples, while CTC^high/ER- samples had higher incidence of alterations in SMAD4, PIK3CA, BRAF and CDK4 compared to the other 2 groups (Table 1). CTC^high/ER- had also higher mutant allele frequency compared to CTC^high/ER+ and CTC^low (MAF. 3% in 73% vs 54% and 31%, respectively, p, 0.001). CTC^low samples had overall lower cfDNA alteration incidence. Similarly, alterations in the ER pathway were more frequent in samples with CTC^high/ER+, whereas alterations in PI3K, cell cycle and P53 pathways were more common in CTC^high/ER- samples. Alterations in the RTK/RAS/RAF pathway were more common in CTC^high samples (23% and 28% for ER- and ER+ vs 9.9% for CTC^low, p = 0.017). Similar results were observed with a 10% threshold. Conclusions: Distinct cfDNA alterations were identified based on ER expression in CTCs in HR+/HER2- MBC. Integrating CTC enumeration and cfDNA profiling may help elucidate resistance mechanisms, identify actionable targets, and predict benefit from continued CDK4/6 inhibition beyond progression. Research Sponsor: Pfizer; Merck KGaA; CrossRef Funder ID: 10.13039/100009945.