Abstract A35: BRAF inhibition increases exosomal PD-L1 protein expression in melanoma
Authors: Kasumova Gyulnara G. , Shi Alvin , Cintolo-Gonzalez Jessica A. , Chein Isabel , Frederick Dennie T. , Alpatov Roman , Michaud William A. , Plana Deborah , Panka David J. , Corcoran Ryan B. , Flaherty Keith T. , Sullivan Ryan J. , Kellis Manolis , Boland Genevieve M. 
Published: February 2019

Abstract
Background: Modulation of the PD-1/PD-L1 axis in melanoma is of critical importance in both the setting of targeted therapy treatment, as well as in newer trials combining targeted therapy with checkpoint blocking antibodies. BRAF inhibition has been shown to increase PD-L1 expression in melanoma tumors. Exosomes are circulating microvesicles that contain a subtranscriptome and/or subproteome of their cell of origin, including tumor cells and immune cells. We evaluated melanoma exosomal PD-L1 protein expression and the effect of BRAF inhibitor (BRAFi) treatment on exosomal PD-L1 protein levels.
Methods: Melanoma cell lines (BRAFi sensitive [A375] and resistant [RPMI7951; induced resistant A375]) and patient plasma were used for analysis. Exosomal vesicles were isolated using centrifugation. Functional proteomics by reverse phase protein array (RPPA) were performed in cell lines and exosomes. RPPA PD-L1 results were quantified in cells and exosomes treated with control (DMSO) and BRAFi (PLX-4720). Exosomal PD-L1 expression from serial patient plasma with metastatic melanoma were assessed using ELISA prior to and after initiation of anti-PD-1 therapy.
Results: Functional proteomic analysis revealed PD-L1 expression on both BRAFi sensitive and resistant cell lines. Cell line derived exosomes demonstrated enrichment of PD-L1 expression compared to their cell of origin, with significantly higher levels of expression in resistant lines and upon treatment with BRAFi therapy. Exosomes derived from patients with melanoma also revealed pre-treatment and on-treatment PD-L1 expression. Furthermore, consistent with cell line data, in a representative patient treated with BRAFi therapy prior to anti-PD-1 therapy, exosomal PD-L1 protein expression increased dramatically upon treatment with BRAFi and preceded a complete response to anti-PD-1 therapy. All other patients were treated only with checkpoint blockade and the majority demonstrated correlation of PD-L1 protein expression with tumor burden.
Conclusions: These results confirm that exosomes from cell lines and patient samples express PD-L1 that can be serially monitored, and that treatment with BRAF inhibition results in increased PD-L1 levels, which in vitro are persistently elevated in BRAFi resistant cells. We also noted that exosomal PD-L1 protein expression levels tend to correlate with tumor burden in patient samples. Measuring PD-L1 expression may serve as a potential biomarker of tumor burden and as an inducible response to BRAFi therapy that predicts synergism with checkpoint anti-PD-1 therapy.
Citation Format: Gyulnara G. Kasumova, Alvin Shi, Jessica A. Cintolo-Gonzalez, Isabel Chein, Dennie T. Frederick, Roman Alpatov, William A. Michaud, Deborah Plana, David J. Panka, Ryan B. Corcoran, Keith T. Flaherty, Ryan J. Sullivan, Manolis Kellis, Genevieve M. Boland. BRAF inhibition increases exosomal PD-L1 protein expression in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A35.