BackgroundBilateral primary breast cancer (BPBC) is a rare type of breast cancer. Studies on the clinicopathologic and molecular characteristics of BPBC in a metastatic context are very limited.MethodsA total of 574 unselected metastatic breast cancer patients with clinical information were enrolled in our next‐generation sequencing (NGS) database. Patients with BPBC from our NGS database were regarded as the study cohort. In addition, 1467 patients with BPBC and 2874 patients with unilateral breast cancer (UBC) from the Surveillance, Epidemiology, and End Results (SEER) public database were also analyzed to determine the characteristics of BPBC.ResultsAmong the 574 patients enrolled in our NGS database, 20 (3.5%) patients had bilateral disease, comprising 15 (75%) patients with synchronous bilateral disease and 5 (25%) patients with metachronous bilateral disease. Eight patients had bilateral hormone receptor‐positive (HR+)/human epidermal growth factor receptor‐negative (HER2−) tumors, and three had unilateral HR+/HER2− tumors. More HR+/HER2− tumors and lobular components were found in BPBC patients than in UBC patients. The molecular subtype of the metastatic lesions in three patients was inconsistent with either side of the primary lesions, which suggested the importance of rebiopsy. Strong correlations in clinicopathologic features between the left and right tumors in BPBC were exhibited in the SEER database. In our NGS database, only one BPBC patient was found with a pathogenic germline mutation in BRCA2. The top mutated somatic genes in BPBC patients were similar to those in UBC patients, including TP53 (58.8% in BPBC and 60.6% in UBC) and PI3KCA (47.1% in BPBC and 35.9% in UBC).ConclusionsOur study suggested that BPBC may tend to be lobular carcinoma and have the HR+/HER2− subtype. Although our study did not find specific germline and somatic mutations in BPBC, more research is needed for verification.

Background Novel human epidermal growth factor receptor 2 (HER2)-directed antibody–drug conjugates prompt the identification of the HER2-low subtype. However, the biological significance of HER2-low expression in breast cancer is unclear. Methods Clinical and genomic data of 579 metastatic breast cancer patients were reviewed from our next-generation sequencing (NGS) database and genomic analysis of early breast cancer patients from TCGA was also analyzed. Findings First, the clinicopathological characteristics of HER2-low patients were profoundly influenced by HR status and no difference of prognosis was observed between HER2-low and HER2-zero patients when paired by HR status, but notably HER2-low patients showed similar metastatic patterns to HER2-positive patients in the HR-positive (HR+ ) subgroup, with more brain and initial lung metastases and more cases of de novo stage IV breast cancer than HER2-zero patients. Second, among patients with primary HER2-low or HER2-zero tumors, the discordance of HER2 status between primary and metastatic tumors was significant, with 48.4% of patients with HER2-zero primary tumors exhibiting HER2-low phenotype in metastatic tumors in the HR+ subgroup. Third, within HR+ and HR-negative subtypes, HER2-low and HER2-zero tumors showed no substantial differences in mutation alterations and copy number variations. Forth, germline BRCA2 mutations were observed only in HER2-low patients in our NGS database, especially in HR+ HER2-low tumors. Finally, three molecular subtypes based on genomic alterations in HER2-low breast cancer were identified, which provided novel insights into heterogeneity in HER2-low breast cancer. Conclusions After correcting for HR expression, only marginal differences in clinical and molecular phenotypes were determined between HER2-low and HER2-zero breast cancer. Therefore, HER2-low breast cancer is insufficient to be defined as a distinct molecular entity, but rather a heterogenous disease.

e13086 Background: The mechanics of tumor microenvironment, primarily induced by extracellular matrix (ECM), function to regulate cell biological behaviors. However, it is unclear if and how triple negative breast cancer (TNBC) cells adapt their biological characteristics to variable extracellular mechanical forces. Methods: TNBC tissues of different stiffness from patients were harvested for untargeted metabolomics to investigate the potential effects of mechanical forces on metabolism. TNBC cells were cultured within gels of different type I collagen (Col-I) densities, which were the major component of ECM, to simulate the effects of extracellular mechanical forces on cells. The metabolic changes of TNBC cells were also examined by using untargeted metabolomics. TNBC cells bathed in different Col-I densities were injected subcutaneously into the breast pad of nude mice. Ferroptosis indicators were evaluated between different groups of mechanical force both in vitro and in vivo. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were carried out to examine the expression of ferroptosis-related genes and proteins between different groups of mechanical force. Results: Untargeted metabolomics studies showed significant differences in ferroptosis-related metabolites between TNBC tissues with different stiffness, which were also verified in TNBC cells subjected to different extracellular mechanical forces. In vitro experiment suggested that TNBC cells cultured within high Col-I density (strong extracellular forces) exhibited higher levels of lipid peroxidation, reactive oxygen accumulation, mitochondria membrane potential and iron concentration, and lower levels of total glutathione and reduced glutathione concentration, compared with low Col-I density (weak extracellular forces). In addition, mitochondria in high Col-I density group appeared to have volume decrease, fracture and fuzzy cristae. The above results indicated that extracellular mechanical force significantly induced ferroptosis in TNBC. RT-qPCR and western blot of ferroptosis-related genes and protein showed that both mRNA and protein levels of aurora kinase A (AURKA) were significantly decreased in TNBC cells with strong extracellular mechanical force. In addition, overexpression of AURKA inhibited ferroptosis caused by mechanical forces. Furthermore, this cause and effect among mechanical force, AURKA and ferroptosis was also evident in vivo experiments. Conclusions: Our data clarified the role of extracellular mechanical forces in TNBC and showed that the extracellular mechanical forces induced ferroptosis through decreasing the expression of AURKA.