Abstract 2174: Evaluation of extracellular vesicles (EVs) as potential predictors of response to immunotherapy (IO) in triple negative early and advanced breast cancer (BC): preliminary evidence from the IRIS project
Authors: Martini Veronica , Stella Arianna , Ayed Rahma Ben , Branni Carmen , Tassone Andrea , Keivan Nazanin , Saggia Chiara , D'Avanzo Francesca , Rossi Valentina , Platini Francesca , Romaniello Incoronata , Giacobino Elena , Guarneri Valentina , Boldorini Renzo Luciano , Mercalli Francesca , Dodaro Ivan , Gambaro Anna , Pinato David James , Gennari Alessandra 
Published: April 2023

BackgroundEvaluation of extracellular vescicles (EVs) has been identified as a potential predictive biomarker in patients treated with IO. In particular, Glypican-1 (GPC-1), was reported to be abnormally expressed in BC tissues and GPC-1+ EVs resulted significantly higher in BC patients than in healthy controls. Triple negative BC (TNBC) is characterized by poor prognosis, due to the lack of effective treatments; in this context, Immune Checkpoint Inhibitors (ICIs) are regarded as promising agents; however, their impact is not the same as in other cancers, such as melanoma and lung. With these premises, the aim of the IRIS project is to evaluate the prognostic and predictive role of tumor and leukocyte-derived EVs in patients with early and advanced TNBC, treated with ICIs.MethodsTwenty consecutive patients have been enrolled so far: 10 with TN MBC PD-L1+ treated with Ist line Nab-Paclitaxel + Atezolizumab and 10 with high-risk TN early BC treated with neo-adjuvant pembrolizumab (KEYNOTE-522 schedule). In all patients, 5 ml of peripheral blood in Na-Citrate were drawn at baseline. An optimized flow cytometry method was used to identify and subtype circulating EVs. EVs were identified as LCD+/phalloidin−events, falling in the scatter area with physical parameters lower than platelets. CD45, GPC-1, EpCAM and PD-L1 were chosen as markers for EVs characterization. Data acquisition and analyses were performed with flow cytometry (FACS Symphony) and FACSDiva v9.0.2. Volumetric count was applied to calculate EV concentrations. Statistical analysis was performed with GraphPad software, and a non-parametric t-test was utilized for EVs characterization, with a p-value &lt; 0.05.ResultsOverall, a significantly lower concentration of circulating tumor-derived EVs (CD45-EpCAM+GPC-1+) was detected as compared to leukocyte-derived EVs (CD45+): Mean±SD = 95.16 ± 219.3 vs 1623 ± 1582; p&lt;0.0001. Similar results were obtained when PD-L1 expression on EVs was considered, with a lower concentration of CD45-EpCAM+GCP-1+PD-L1+ compared to CD45+PD-L1: Mean ± SD: 12.63 ± 26.97 vs 656.8 ± 1012; p&lt;0.0001. When taking into account the two patient subsets, ie MBC TN and early high risk TN, no significant difference was detected in concentration for circulating leukocytes- and tumor-derived PD-L1+ EVs: Mean ± SD cancer-derived EVs in MBC vs early BC: 6.4 ± 11.19 vs 19.56 ± 37.22, p=0.68; leukocytes-derived EVs in MBC vs early BC: 924.8 ± 1316 vs 359 ± 405, p=0.97. ConclusionThese preliminary data demonstrate that tumor-derived PD-L1+ EVs can be detected in advanced as well as in early TNBC and may represent a novel biomarker to assess response to ICIs. Patient recruitment and Flow Cytometry analyses are ongoing to confirm these data and to assess their predictive/prognostic role in TNBC.

Citation Format: Veronica Martini, Arianna Stella, Rahma Ben Ayed, Carmen Branni, Andrea Tassone, Nazanin Keivan, Chiara Saggia, Francesca D'Avanzo, Valentina Rossi, Francesca Platini, Incoronata Romaniello, Elena Giacobino, Valentina Guarneri, Renzo Luciano Boldorini, Francesca Mercalli, Ivan Dodaro, Anna Gambaro, David James Pinato, Alessandra Gennari. Evaluation of extracellular vesicles (EVs) as potential predictors of response to immunotherapy (IO) in triple negative early and advanced breast cancer (BC): preliminary evidence from the IRIS project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2174.