mutations are biomarkers in breast cancer patients who develop metastatic disease after endocrine therapy (ET). Recently, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) have approved Elacestrant, a selective estrogen receptor degrader for patients harboring mutations. This has necessitated the establishment of reliable and sensitive NGS- or PCR-based assays to detect these resistance mutations in liquid biopsy samples.

We evaluated NGS results of a pan-cancer cohort of almost 6000 patients from two major German institutes of pathology, to show that the occurrence of mutations is extremely rare (<1%) in ET-naïve patients. This suggests that mutations arise almost exclusively under the pressure of ET. Therefore, we designed a breast cancer-specific hybrid capture-based NGS liquid biopsy assay covering 12 breast cancer-related genes, including , , , , , and . We validated the HS2-Mamma-LIQ assay extensively using reference material to detect mutations to 0.1% variant allele frequency (VAF) and compared the performance to a commercially available ddPCR assay.

We show the results of routine diagnostic analysis of the first consecutive 354 patients with activating mutations rate of 43%, with 20% of patients harboring co-mutations in and other genes underlining the relevance of tumor heterogeneity. Our study highlights liquid biopsy as a preferred approach for monitoring mutations in breast cancer patients by showing cases where NGS analysis suggests complex tumor heterogeneity with multiple as well as mutations at different VAFs.

Our findings not only corroborate prior research concerning the rarity of these mutations in unselected patients but also emphasize the importance of robust and broad molecular assays rather than single gene assays in their detection and characterization in the diagnostic setting. Advantages of different approaches are discussed to address the current clinical need.