Gene expression analysis in circulating tumour cells to determine resistance to CDK4/6 inhibitors plus endocrine therapy in HR + /HER2- metastatic breast cancer patients.

BACKGROUND: Metastatic breast cancer (BC) is the main cause of cancer-related mortality in women worldwide. HR + /HER2- BC patients are treated with endocrine therapy (ET), but therapeutic resistance is common. The combination of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) with ET was approved for metastatic BC patients and extended the median progression-free survival to 24 months. This therapy is not always effective, and in every patient, resistance ultimately occurs, but the underlying resistance mechanisms remain unclear. To address this gap, we explored circulating tumour cells (CTCs) as biomarkers to assess treatment response and resistance in metastatic HR + /HER2- BC patients receiving CDK4/6i plus ET. METHODS: In total, 53 HR + /HER2- metastatic BC patients who received a CDK4/6i plus ET as first-line treatment were analysed, including samples from internal and external validation cohorts. CTCs were isolated using the negative enrichment approach RosetteSep (STEMCELL Technologies) or positive immunomagnetic selection targeting EpCAM, EGFR, and HER2 (AdnaTest EMT-2/StemCell Select™, QIAGEN). RNA was extracted from CTCs and PBMCs for nCounter analysis (Pancancer pathways panel) in a discovery phase. Subsequent validation was performed by RT-qPCR. RESULTS: CTC gene expression analysis revealed that non responder patients (those who experienced disease progression before 180 days) exhibited elevated PRKCB (p-value: 0.011), MAPK3 (p-value: 0.006) and STAT3 (p-value: 0.008) expression, while responders showed increased CDK6 (p-value: 0.011) and CCND1 (p-value: 0.035) expression at baseline. CTC transcriptional characterization revealed a gene expression signature (STAT3(high)PRKCB(high)CDK6(low)) that accurately classified HR + /HER2- metastatic BC patients who responded to CDK4/6i plus ET, regardless of the CTC isolation method (AUC > 0.8). CTC characterization at progression also identified biomarkers linked to therapy resistance, including the epigenetic regulators EZH2 and HDAC6 and the cell cycle regulator CDC7, which could guide the selection of subsequent therapy lines. The expression of the CDK4 and STAT3 genes in CTCs was associated with progression-free survival and overall survival, respectively. Likewise, the presence of ≥ one CTC after one cycle of therapy predicts a worse prognosis. CONCLUSIONS: CTC gene expression provides information about treatment outcomes in HR + /HER2- metastatic BC patients receiving CDK4/6i plus ET and could guide personalized strategies and improve prognosis.