Elucidating PI3K / AKT / PTEN Pathway Alterations at Single‐Cell Level in CTCs From HR +/ HER2 − Metastatic Breast Cancer

Alterations in the PI3K signaling pathway are common in hormone receptor-positive (HR+)/HER2-negative metastatic breast cancer (MBC) and are associated with response to PI3K and AKT inhibitor-targeted therapies. When metastasis biopsy is unavailable for genetic testing, circulating tumor DNA (ctDNA) analysis is an alternative strategy for tumor genotyping, but it is less sensitive for gain and loss analysis. Here, we assess molecular alterations of the genes of this axis-PIK3CA, AKT1, and PTEN-in circulating tumor cells (CTCs) from patients with HR+/HER2- MBC. CTCs from blood were isolated as single cells with DEPArrayNxT. Libraries were prepared for copy number analysis (CNA) and investigation of PIK3CA, AKT1, and PTEN mutational status. A subset of viable CTCs underwent 3' RNA sequencing for transcriptomic profiling. CTCs of 4/5 MBC patients (80%) exhibited single nucleotide variants (SNVs), and heterogeneity was observed at inter- and intra-patient levels. CNA profiling of CTCs from 8 patients revealed amplifications and gains of chromosomal regions hosting PIK3CA, AKT1, and PTEN. Notably, one patient presented both 10q32.31 loss and a nonsense mutation of the remaining PTEN allele, suggesting PTEN loss of function, an event potentially missed by ctDNA analysis. Integrated transcriptomic analysis highlighted patient-specific enrichment of pathways related to metabolism and RNA processing. Conclusively, single-CTC analysis enables comprehensive characterization of the PI3K/AKT/PTEN pathway, revealing molecular heterogeneity and capturing events not detectable by tumor tissue or ctDNA. This approach may enhance molecular stratification and guide the use of targeted therapies in MBC, supporting its integration into future precision oncology frameworks.