INTRODUCTION: Trastuzumab (TZM) resistance remains a primary cause of mortality in HER2-positive breast cancer (BC) patients. We hypothesize that this resistance may be partially due to synergistic interactions among members of the HER oncogene family, compensating for inhibition when one member is targeted. Previously, we developed an MLPA-based tool to assess CNV of the four HER oncogenes. Our current objective is to investigate the association between co-amplification of HERs and resistance to TZM. OBJECTIVE: To analyze the correlation between CNV/expression of the four HER oncogenes and clinical variables in HER2-positive BC patients using both clinical and computational approaches. RESULTS: Clinical study: DNA was extracted from FFPE tissue of 49 HER2-positive patients. Using our MLPA X026 probemix (MCR Holland), we confirmed 42/49 tumors as HER2-positive. Co-amplification of HER2 with at least one other HER oncogene was observed in 12/42 samples (29%). Among 42 patients, 6 (14.3%) experienced disease recurrence; notably, 1/6 was HER2-negative (refractory to TZM), while all the remaining 5 patients exhibited HERs co-amplification (p=0.001). Significant correlations were found between HER2/EGFR and HER2/HER3 co-amplification and recurrence (p<0.001 and 0.03). In silico study: Analysis of TCGA data from 67 HER2-positive BC patients (PAM50RNAseq) revealed that high co-expression of HER2/HER3 significantly correlated with decreased overall survival (<0.0001). Differential gene expression analysis indicated downregulation of tumor microenvironment (TME)-related genes in these tumors. This altered TME affects immune cell populations such as NK, macrophages, and CD8 cells, along with reduced INF and cytokine signatures (p<0.05). CONCLUSION: Co-amplifications of HER2 with HER3 reduce OS in HER2-positive BC patients, potentially influencing the tumor microenvironment and antitumor response. Further studies are needed to elucidate how HERs “work together for the family".