• ctDNA screening phase:

• Main inclusion criteria:

• • Female (both pre- and postmenopausal) or male patients with histologically confirmed ER positive (regardless of PR),

• HER2 negative breast cancer, according to local pathologist:

⁃ ER-positive defined as ≥ 10% of cells staining positive for ER or Allred proportion score ≥3

⁃ HER2-negative defined as a score of 0, 1+ by immunohistochemistry (IHC) or a negative in situ hybridization (ISH) based on single-probe average HER2 copy number, as per American Society of Clinical Oncology guidelines

⁃ Intermediate to high risk of recurrence after definitive treatment for early breast cancer, defined as:

• FOR PATIENTS TREATED WITH PRIMARY SURGERY:

⁃ Any patient with ≥ 4 positive axillary lymph nodes (stage pN2-3).

⁃ 1-3 positive axillary lymph nodes (stage pN1) and either:

⁃ Tumour size ≥ 5 cm or/and

⁃ Histologic grade 3 or/and

⁃ Ki67≥20% or/and

⁃ High genomic risk defined as Oncotype Dx Recurrence Score \>=26, Mammaprint high risk, Prosigna score \>40 or EPclin risk score \>=4.0.

⁃ Negative axillary lymph nodes (stage pN0) and tumour size ≥ 5 cm and either

⁃ Histologic grade 3 a or/and

⁃ Ki67≥20% and/or

⁃ High genomic risk defined as Oncotype Dx Recurrence Score \>=26, Mammaprint high risk, Prosigna score \>60 or EPclin risk score \>=4.0. FOR PATIENTS TREATED WITH NEOADJUVANT

• SYSTEMIC TREATMENT FOLLOWED BY SURGERY:

⁃ Patient may have received neoadjuvant endocrine therapy or neoadjuvant chemotherapy provided that:

⁃ The initial tumour and/or the tumour after surgery meet the criteria above defined for patients treated with primary surgery or the initial tumour was staged as cT4anyN and

⁃ There is no pathological complete response, defined as no invasive disease in the breast and axilla (ypT0/is ypN0).

⁃ Age ≥18 years

⁃ Patients must have received at least 1 year and up to 7.5 years of ET and planned to continue adjuvant ET during ctDNA screening phase

⁃ Previous adjuvant CDK4/6 inhibitor or PARP-inhibitor treatment is allowed provided it is completed

⁃ Invasive multicentric / multifocal disease is allowed provided that all the tested foci are ER+ HER2-. A sample from the highest-risk one, according to the investigator decision based on the size and grade, should be sent to Natera to build the patient ctDNA assay.

⁃ Available tumour sample from resected or biopsied tissue, with a tumour content of ≥20% (30% preferred) either before or after macro dissection (if performed) and a cell viability of a minimum 100 cells.

⁃ Core Needle Biopsies (CNB): recommended minimum of four (4) cores per block

⁃ Fine Needle Aspirates (FNA) are not accepted

⁃ The following sample types are acceptable:

⁃ 6-10 unstained slides (charged and unbaked) of 10μm each (or 12-19 unstained slides at 5 μm each), PLUS one contiguous H\&E slide. Minimum total tissue thickness must be 60μm OR

⁃ FFPE tissue block with 25mm2 minimum surface area

⁃ Written informed consent must be given according to ICH/GCP, and national/local regulations.