This study retrospectively analyze tumor samples and clinical data from Greek women with operable breast cancer of intermediate or high-risk of relapse who participated in HeCOG adjuvant studies. Patients were treated with dose-dense sequential adjuvant chemotherapy regimens mainly including epirubicin, cyclophosphamide, taxanes, and "intensified" CMF.

The main goal is to investigate how tumor characteristics and genetic mutations relate to breast cancer subtype and patient outcome (OS). Tumor samples were centrally reviewed using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS). Tumors were classified into molecular subtypes such as: Luminal A, Luminal B, Luminal-HER2, HER2-enriched and Triple-negative.

Important biomarkers analyzed included: ER (estrogen receptor), PgR (progesterone receptor), HER2, Ki67 (cell proliferation marker), EGFR and CK5 (basal-like markers), CD8-positive tumor infiltrating lymphocytes (immune response marker) as well as recurrent alterations in genes commonly mutated in breast cancer including among others TP53, PIK3CA, GATA3, CDH1, PTEN and AKT1. HER2 and TOP2A gene amplification were assessed using FISH.

Tumor immune infiltration (TILs) was also evaluated by expert pathologists. DNA extracted from archived tumor tissue was analyzed with a custom breast cancer NGS panel targeting genes commonly mutated in breast cancer.

Inclusion Criteria:

• Women with histologically confirmed epithelial breast cancer
• Age >18 years
• Pathological stage
• Eastern Cooperative Oncology Group performance status 0-1
• Normal cardiac function and adequate bone marrow, hepatic and renal function.

This study includes tumors from patients with operable breast cancer of intermediate or high risk of relapse, according to St.Gallen criteria, treated with dose-dense sequential adjuvant chemotherapy with epirubicin, cyclophosphamide, taxane (only a certain group did not include a taxane) and "intensified" CMF.