Introduction: CDK4/6 inhibitors (CDK4/6i), such as palbociclib, combined with endocrine therapy, are first line therapies for advanced breast cancer (BrCa). CDK46i are well tolerated but not curative and patients often experience resistance. Several studies have suggested molecular features of resistance to CDK4/6i. However, they are based on small cohorts and thus limit the ability to define precision medicine opportunities for resistant patients.

Results: We assembled and analyzed a multi-modal real-world cohort of 1,248 breast cancer patients, enriched for pre- and post-CDK4/6i, to derive a comprehensive molecular landscape of CDK4/6i resistance. We confirmed known and identified novel genetic aberrations enriched in post-CDK4/6i including ESR1 mutations and RB1 loss (64% patients); however, 36% of patients lack genomic aberrations linked to resistance. We additionally analyzed longitudinal immunohistochemistry (IHC) (N=432) and RNA sequencing (N=25) data and found widespread subtype transitions upon CDK4/6i treatment. This included transition from HR+/Her2- to basal (7%), HR+/Her2+ (10%), and HR-/Her2+ (1%). Spatial transcriptomics data revealed HR+ tumors comprise of LumA, LumB and Her2 cells co-localized within tumor regions. Furthermore, longitudinal bulk RNA-Seq confirmed IHC results and additionally identified LumA to LumB transition post-CDK4/6i. These suggest that the expansion/emergence of aggressive subtype represents a major mechanism of CDK4/6i resistance. Lastly, genomic and transcriptomic integrative clustering identified CDK4/6i-resistant patient segments with distinct molecular features including somatic mutations, transcriptional subtypes, and transitions. Two segments showed strong enrichment of post-CDK4/6i tumors and biomarkers of resistance. Including one enriched in with ESR1 mutations, LumB tumors, and another enriched in tumors which post-CDK4/6i have transitioned into either HR+/Her2+ tumors (with ERBB2 mutations), or TNBC (with LAR-like phenotype) basal tumors.

Discussion: Our study provides a comprehensive analysis of the genomic and phenotypic changes associated with resistance to CDK4/6i in large RW breast cancer cohort. The identification of distinct resistance segments highlights the heterogeneity and complexity of mechanisms driving patient resistance. This provides a resource for further understanding and development of personalized treatment strategies to overcome CDK4/6i resistance.