Loss of HER2 after HER2-targeted treatment Abstract Purpose HER2 expression has been reported to be discordant between primary tumor and metastatic tissue. Patients and methods HER2 discordance and relation to HER2-targeted treatment was investigated in 227 patients with primary breast cancer. Results HER2 discordance between primary biopsy and second biopsy after neoadjuvant or adjuvant treatment was observed in 20.7%. This discordance was related only to the use of HER2-targeted treatment: 30 of 33 (90.9%) women with downgraded HER2 expression underwent a HER2-targeted therapy, whereas in the group of patients with concordant HER2 expression, only 32 of 180 (17.8%) received HER2-targeted treatment (p < 0.0001). HER2 discordance was associated with reduced disease-free survival but not overall survival. In a second cohort, including patients with HER2 overexpressing tumors, trastuzumab treatment was associated with change of HER2 expression from positive to negative in 47.3% of cases. Addition of pertuzumab increased the rate of HER2 loss up to 63.2%. Notably, the interval between last HER2-targeted treatment and the time of surgical excision of the tumor after neoadjuvant chemotherapy (NACT) or the biopsy of the metachronous metastasis was associated with a significant change in HER2 expression. The median time between NACT and the time of surgical excision was 23 days (range 5–81 days) for tumors with decreased HER2 expression and 51 days (range 10–179 days) for tumors with concordant HER2 expression. Furthermore, median time between the end of adjuvant treatment and second histology of the metachronous metastases accounted for 15 days (range 2–165 days) and 478 days (range 7–2739 days) was observed in the group of patients with decreased or unchanged HER2 expression, respectively. Conclusion The interval between anti-HER2 treatment and the determination of HER2 in second histology is strongly associated with HER2 expression.