Genomic and transcriptomic changes associated with tucatinib exposure in HER2-positive breast cancer brain metastases

Background: In the HER2CLIMB trial, the addition of HER2-directed tyrosine kinase inhibitor tucatinib to trastuzumab and capecitabine improved progression-free survival (PFS) in patients with HER2-positive (HER2+) breast cancer brain metastases, with 1-year PFS of 24.9% on tucatinib versus 0% on placebo. While prior studies demonstrated genomic divergence between brain metastases and primary tumors, the molecular effects of tucatinib in the brain remain poorly characterized, in part due to limited availability of resected brain metastasis tissue. We compared genomic and transcriptomic features of HER2+ breast cancer brain metastases with and without tucatinib exposure to identify candidate molecular adaptations potentially relevant to tucatinib resistance. Methods: Brain metastases specimens were obtained from 17 patients with HER2+ breast cancer undergoing neurosurgical resection. All patients consented to donate tissue on an IRB-approved protocol. After quality control, ribosomal RNA-depleted RNA sequencing was performed on 10 tumors: 5 tumors that progressed after tucatinib treatment and 5 tucatinib-unexposed. Differential gene expression analysis was adjusted for hormone receptor (HR) status of brain metastases. Thirteen tumors were analyzed by Whole Exome Sequencing (WES): 7 tumors that progressed after tucatinib and 6 tucatinib-unexposed. Matched normal tissue was available for all samples for WES analyses. Somatic variants were annotated for predicted deleterious functional impact using SIFT and PolyPhen-2 software, as well as data from NCBI ClinVar and the “impact” metric from the Ensembl Variant Effect Predictor, and a variant allele frequency (VAF) threshold of > 10 % was applied for global analyses. Results: After adjustment for HR status using the RNAseq data, 127 genes were differentially expressed between tucatinib-exposed and unexposed tumors. Tumors progressing on tucatinib showed enrichment of transcriptional programs related to stemness, epithelial-mesenchymal transition (EMT), and metabolic reprogramming. No transcriptional enrichment of established HER2 resistance pathways, including MAPK or PI3K/AKT signaling, was observed. Genomic profiling identified 17 genes with the highest number of activating mutations at VAF≥10% after tucatinib exposure that were predominantly involved in proliferation, migration, immune evasion and cancer stemness. No ERBB2 gatekeeper mutations (T798M, T798I) or common HER2 driver mutations (V777L) were detected at VAF≥10% after tucatinib exposure. Instead, tucatinib-exposed tumors more frequently harbored potentially deleterious mutations in ERBB family members (ERBB3 and ERBB4), ERBIN, and MET, whereas ERBB2 mutations, when present, occurred at low allelic frequency (<5%). Conclusion: In this exploratory cohort of resected HER2+ breast cancer brain metastases, tucatinib exposure was associated with genomic and transcriptomic patterns consistent with adaptive remodeling rather than classic HER2 resistance mutations. Alterations within the broader ERBB signaling network and activation of stemness / EMT-associated transcriptional programs may contribute to tucatinib resistance in the brain and warrant further investigation in larger longitudinal datasets.