Abstract B58: Detection of ESR1 gene fusions in breast cancer cell-derived exosomal RNA

Background: Estrogen receptor-positive (ER+) breast cancer is often intractable due to endocrine therapy resistance. Although ER basepair hotspot mutations have been well characterized in ~20-30% of endocrine-resistant disease, accumulating evidence from our group and others suggests that other genetic alterations such as ER fusions (e.g., ESR1-DAB2) could also play a role. As an important component of liquid biopsies, exosomes have recently gained much attention in the field of clinical oncology. It is well established that some species of RNA, typically microRNA and mRNA, are packaged into these small membrane-enclosed structures, which could potentially serve as a noninvasive biomarker to constantly and comprehensively monitor tumor progression and tumor cell evolution. We tested whether ESR1 fusions could be detected in exosomal RNA (exoRNA) produced by breast cancer cell lines. Methods: Multiple methods were used to characterize exosomes isolated from cell culture media: 1) Nanoparticle tracking analysis (NTA) confirmed concentration and size of exosomes, 2) immunoblotting identified protein markers that are enriched or lacking in exosomes, 3) transmission electron microscopy validated the size and shape of exosomes. During exoRNA extraction, DNA contamination was removed by DNase treatment. Fusion-specific regions in exoRNA were amplified by standard PCR. Results: We used breast cancer cell lines stably infected with cDNA encoding ESR1 fusions ESR1-DAB2 and ESR1-SOX9. NTA shows that our isolated exosomes had a size ranging from 30-150 nm, with the peak at 80 nm. Immunoblotting showed TSG101 and CD81 as positive markers of our exosomes, while Grp94 was negative. By RT-PCR, we identified a fusion-specific region amplified for both ESR1-SOX9 and ESR1-LPP from exoRNA for the fusion cell lines. Conclusions: Collectively, these data indicate that multiple ESR1 fusions are detectable in breast cancer cell line-derived exoRNA. Citation Format: Tiantong Liu, Jagmohan Hooda, Nils Ludwig, Theresa Whiteside, Jennifer M. Atkinson, Steffi Oesterreich, Adrian Lee. Detection of ESR1 gene fusions in breast cancer cell-derived exosomal RNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B58.