Breast cancer is the most common type of cancer as well as the first cause of cancer-specific mortality among women worldwide (Arnold et al., 2022). It is, however, not a single disease but rather a group of distinct diseases that arise from the same organ. Human Eidermal growth factor Receptor 2 (HER2) is an orphan tyrosine kinase receptor of the HER family that is over-expressed in 15% of breast cancer (Slamon et al., 1987). HER2 over-expression is the result of gene amplification in 95% of cases (Marchiò & Reis-Filho, 2008). Initially considered a negative prognostic factor, HER2 represents a target for individualized therapy as it is both a tumor driver and an example of oncogene addiction (Baselga & Mendelsohn, 1994). The introduction of HER2-targeted therapies, among which trastuzumab is the forerunner, has indeed dramatically changed the natural history of this disease (reviewed by Swain, Shastry, & Hamilton, 2022).

HER2 status of all newly diagnosed breast cancer is routinely assessed in primary tumor according to the American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) guidelines (Wolff et al., 2018), including immunohistochemistry (IHC) of HER2 protein (p185) expression and in situ hybridization (ISH) analysis of ERBB2 gene copy number. Briefly, cases with complete membrane staining demonstrating strong intensity in more than 10% of tumor cells are classified as HER2 IHC positive (3+), cases with weak to moderate complete membrane staining in more than 10% of tumor cells HER2 IHC equivocal (2+), cases with incomplete membrane staining barely perceptible and in more than 10% of tumor cells are classified as HER2 IHC weak (1+), and cases with no staining or incomplete membrane staining barely perceptible and in less than 10% of tumor cells are classified as HER2 IHC negative. ERBB2 amplification is evaluated as a ratio between copies of the ERBB2 gene, which is located on chromosome 17, and copies of the CEP17 reference gene representing chromosome 17 centromere (number ERBB2 copies/number of CEP17 copies>2.2) or an absolute number of ERBB2 copies per cell>6. Guidelines for HER2 ISH interpretation on tissue sections require counting of the signal in at least 20 cells in the area of strongest signal intensity. In current clinical practice, HER2 IHC is always performed, followed by ISH only in cases with 2+ IHC staining intensity, and HER2 3+ by IHC or 2+ by IHC and ISH amplified cases are considered eligible for HER2-targeted therapy (Denduluri et al., 2021, Giordano et al., 2022).

Overall, HER2 status seems to be conserved as breast cancer metastasizes (Khasraw, Brogi, & Seidman, 2011). However, one in ten patients with breast cancer eventually modifies their initial HER2 status during tumor progression (Schrijver et al., 2018). The variation in HER2 status between the primary tumor and paired metastasis within a patient is usually referred to as ‘receptor conversion’, and occurs twice as often from positive to negative than vice versa. Several studies have shown improved outcomes in patients without HER2 conversion as compared to those with HER2 conversion (Chang et al., 2011, Edgerton et al., 2003), and in patients with HER2 conversion from negative to positive rather than the opposite (Lower, Glass, Blau, & Harman, 2009) especially when treated with targeted therapy (Pizzuti et al., 2021). It is still debated whether HER2 conversion is due to primary tumor heterogeneity (Ferrari et al., 2016), interpretative difficulties at initial diagnosis (Tapia, Savic, & Wagner, 2007), or rather tumor biology (Lipton et al., 2005, Swanton, 2012). As change in the phenotype may mean the possibility to add a potentially effective therapeutic option (Amir et al., 2012), HER2 reassessment in metastatic lesions should be carefully taken into account, whenever feasible (Gennari et al., 2021, Van Poznak et al., 2015).

Biopsies from most metastatic sites can be obtained with the use of imaging and interventional radiology on a routine basis. However, these techniques are invasive and may lead to discomfort or complications. Therefore, the use of circulating biomarkers to circumvent these issues is desirable (reviewed by Alix-Panabières, Schwarzenbach, & Pantel, 2012) and the potential clinical utility of ctDNA and CTCs in breast cancer is being addressed (Bidard et al., 2021, Bidard et al., 2022, Turner et al., 2023).

In this regard, the analysis of circulating tumor cell (CTC) and cell-free circulating tumor DNA (ctDNA) has the potential to assess HER2 status during breast cancer disease trajectory, as well as to clarify the biological basis of HER2 discrepant cases.

Herein, we review the relevant literature on current methods for testing HER2 in the CTCs and ctDNA, clinical trials evaluating their prognostic and predictive value in breast cancer patients, as well as recent advances in the field. We also discuss potential causes of HER2 discordance between primary tumor, circulating biomarkers and recurrent disease with a special focus on technical aspects.