Deep proteomic analysis of plasma exosomes in patients with advanced, hormone receptor-positive breast cancer treated with palbociclib and tamoxifen.
Authors: Ma Xiuyuan , Gao Yu , Mohapatra Gayatry , Coleman Louis , Simons Yael , Truica Cristina I. , Blaes Anne Hudson , Rana Jatin , Tandra Pavankumar , Green Lauren , Liu Li C , Yu Menggang , Toppmeyer Deborah , O'Regan Ruth , Wisinski Kari Braun , Danciu Oana Cristina , Hoskins Kent ,   Big Ten Cancer Research Consortium
Published: June 2021

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Background: Combining a CDK4/6 inhibitor (CDK4/6i) with endocrine therapy (ET) in advanced, hormone receptor (HR)-positive, HER2-negative breast cancer (BC) doubles median progression-free survival, but eventually drug resistance and disease progression occur. For most patients, the mechanism of resistance is unknown. Exosomes are membrane-bound extracellular vesicles that contain lipids, proteins, and nucleic acids, and are released from tumors as a form of intercellular communication. Exosomes can be recovered from plasma, and analysis of their cargo provides a dynamic read-out of biological pathways that are activated in cancer cells. Proteomic analysis of plasma exosomes may provide insight into mechanisms of resistance that emerge during treatment with CDK4/6i-ET. Methods: The Big Ten Cancer Research Consortium conducted a single arm, phase II trial of palbociclib plus tamoxifen as first line therapy for advanced, HR+/HER2- BC (NCT02668666). Whole blood was collected in Streck tubes from study participants (n = 49) at baseline, at disease progression, and at time points during study treatment. Plasma was separated and stored at -80C within 48 hours of collection. Exosomes were isolated from thawed plasma using commercially available kits and ultracentrifugation. Exosome extraction and purification was optimized for protein recovery. Purified exosomes were processed for proteomic analysis and labeled with TMT10 (tandem mass tag 10plex) and quantified with the QExactive HF mass spectrometer. Ultrasensitive mass spectrometry provided deep proteomic coverage of exosomal proteins and detected various post-translational modifications (PTM). Data were analyzed with a pipeline developed in our lab using an improved SEQUEST/ProLuCID database search engine and Percolator data filtering toolchain. Exosome protein expression was determined at baseline, at best response and at the time of progression. Results: With our ultrasensitive proteomic method, we detected more than 500 exosome proteins from as little as 100 ng of purified exosomes. A significant enrichment of exosome specific markers was observed when comparing patient samples with healthy donor samples. Enrichment of surface glycoproteins (e.g. CD44) was seen in BC patient samples, as in previous reports. Ultrasensitive proteomics also detected PTM including phosphorylation, methylation, oxidation, deamidation, and glycosylation. Differential proteomic and PTM profiles comparing samples collected from responding patients at baseline vs. at progression will be presented. Conclusions: Our innovative method provided an unparalleled portrait of the proteomic landscape of plasma exosomes during treatment with CDK4/6i-ET. This powerful approach may provide novel insights into mechanisms of resistance that emerge during treatment. This study was funded by Pfizer. Clinical trial information: NCT02668666 .