Abstract Background: Patients with metastatic breast cancer (MBC) often have short survival, and clinical disease management is challenging due to tumor heterogeneity, treatment resistance, and unique contributions of organ-specific metastatic microenvironments. Methods: The retrospective and prospective phases of the AURORA US Metastasis Project have profiled samples from 220 MBC patients using RNA-seq, DNA-seq, and DNA methylation arrays. RNA-seq has been performed on 392 specimens from 143 primary tumors and 249 metastases. In total, there are 162 primary-metastasis pairs from 134 patients (13 patients have 2+ metastases profiled). The 249 metastatic samples include 116 liver, 29 lymph node, 17 lung, 14 bone, 10 brain, and 8 skin metastases. Each specimen was assayed to determine its clinical and molecular subtypes. Here we investigate the molecular causes of clinical and PAM50 subtype switching in primary tumor-metastasis pairs using multi-platform data. Results: Of the patients with paired data in the AURORA dataset, 38/134 (28%) had clinical subtype (HR+/HER2+, HR-/HER2+, HR+/HER2-, TNBC) switches between the primary tumor and paired metastasis. The most common clinical subtype switch was loss of hormone receptor expression (16/38 patients) in the metastatic tumor. Many of these observations have been confirmed by concomitant RNA and/or DNA changes. In addition, 59/134 (44%) patients had PAM50 molecular subtype switches. 26/59 (44%) patients had a Luminal A primary tumor which switched either into Luminal B (16/26) or HER2-Enriched (7/26) subtypes, suggesting recurrent progression mechanisms during metastasis. For example, patient ARRP had a Luminal A primary tumor and a HER2-Enriched bone metastasis. Using DNA clonality data, we identified a clonal mutation in the DNA binding domain of FOXA1 (H247L) as well as mutations in CDH1 (frameshift) and NLRC3 (Q650H) that originated in the primary tumor. A distinct, dominant clone arose in the metastasis, harboring oncogenic activating mutations in PIK3CA (H1047R) and ERBB2 (L869R). There was also an increase in RNA expression of ERBB2 and FGFR4 but no change in ERBB2 copy number. This is in alignment with previous publications demonstrating a shift to the HER2-Enriched subtype, often without gain of HER2 amplification. Interestingly, there were 21 patients that had both clinical and PAM50 subtype switches. Patient ANFC had a HR+/HER2- primary tumor (Luminal A) and a TNBC liver metastasis (HER2-Enriched). Notably, the metastasis lost clinical expression of ER and PR (by IHC), and this was also observed through a decrease in ESR1 and PGR mRNA expression. Using DNA clonality data, we identified 5 new clones in the metastasis that were not present in the primary tumor. Finally, we observed that basal-like primary tumors with a PAM50 subtype switch in the paired metastasis had a significantly lower correlation to the basal-like centroid compared to primary tumors with a basal-like paired metastasis (p=0.0053). This suggests that either these tumors had significant intra-tumoral heterogeneity in the primary tumor and the metastasis originated from a non-basal-like portion of the tumor, or the switches from a basal-like primary tumor are a byproduct of the PAM50 bioinformatics algorithm (i.e. the tumor is very close to 2 subtype centroids). Ongoing analyses will precisely determine which subtype switches can be attributed to genetic or epigenetic causes, organ site biases, and/or bioinformatic methodologies. Conclusions: Using multi-platform molecular analyses, we identified cases of clinical and molecular subtype switching in primary-metastasis pairs. These findings begin to elucidate the underlying molecular drivers of tumor progression and subtype switching in patients with MBC, which may have implications for clinical treatment. Citation Format: A. R. Michmerhuizen, B. M. Felsheim, G. L. Wheeler, B. J. Kelly, M. Singha, T. Hinoue, W. C. Nenad, A. C. Garrido-Castro, J. M. Balko, K. Giridhar, U. Chandran, A. V. Lee, L. A. Carey, T. A. King, K. A. Hoadley, P. W. Laird, E. R. Mardis, C. M. Perou, AURORA US Metastasis Network. Molecular causes of subtype switching in primary and metastatic breast tumor pairs from the AURORA US Project abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PD2-06.
Michmerhuizen et al. (Tue,) studied this question.