Clinical relevance of the combined analysis of circulating tumor cells and anti-tumor T-cell immunity in metastatic breast cancer patients.

Background: Metastatic breast cancer (mBC) is a heterogeneous disease with varying responses to treatments and clinical outcomes, still requiring the identification of reliable predictive biomarkers. In this context, liquid biopsy has emerged as a powerful tool to assess in real-time the evolving landscape of cancer, which is both orchestrated by the metastatic process and immune-surveillance mechanisms. Thus, we investigated circulating tumor cells (CTCs) coupled with peripheral T-cell immunity to uncover their potential clinical relevance in mBC. Methods: A cohort of 20 mBC patients was evaluated, before and one month after starting therapy, through the following liquid biopsy approaches: CTCs enumerated by a metabolism-based assay, T-cell responses against tumor-associated antigens (TAA) characterized by interferon-γ enzyme-linked immunosorbent spot (ELISpot), and the T-cell receptor (TCR) repertoire investigated by a targeted next-generation sequencing technique. TCR repertoire features were characterized by the Morisita's overlap and the Productive Simpson Clonality indexes, and the TCR richness. Differences between groups were calculated by Fisher's, Mann-Whitney or Kruskal-Wallis test, as appropriate. Prognostic data analysis was estimated by Kaplan-Meier method. Results: Stratifying patients for their prognostic level of 6 CTCs before therapy, TAA specific T-cell responses were detected only in patients with a low CTC level. By analyzing the TCR repertoire, the highest TCR clonality was observed in the case of CTCs under the cut-off and a positive ELISpot response (p=0.03). Whereas, at follow-up, patients showing a good clinical response coupled with a low number of CTCs were characterized by the most elevated TCR clonality (p<0.05). The detection of CTCs≥6 in at least one time-point was associated with a lower TCR clonality (p=0.02). Intriguingly, by combining overall survival analysis with TCR repertoire, we highlighted a potential prognostic role of the TCR clonality measured at follow-up (p=0.03). Conclusion: These data, whether validated in a larger cohort of patients, suggest that the combined analysis of CTCs and circulating anti-tumor T-cell immunity could represent a valuable immune-oncological biomarker for the liquid biopsy field. The clinical application of this promising tool could improve the management of mBC patients, especially in the setting of immunotherapy, a rising approach for BC treatment requiring reliable predictive biomarkers.