Abstract 5013: Toward better selection of PD-1 blockade responders through direct markers of adaptive immune resistance
Authors: Shin Daniel S. , Garcia-Diaz Angel , Escuin-Ordinas Helena , Palaskas Nicolaos J. , Komenan Sara M. , Graeber Thomas G. , Comin-Anduix Begonya , Ribas Antoni 
Published: August 2015

Abstract
Introduction. The role of PD-L1 as a predictive biomarker for response to PD-1 blockade is unclear. Emerging evidence, including our findings reported here, suggests that PD-L1 expression is important only when induced by the presence of T cells producing interferons, in what is termed adaptive immune resistance (Tumeh, Nature 2014). Therefore, studying PD-L1 expression in response or not to interferons may define molecular mechanisms to select patients whose tumors are incapable to respond to T cell infiltration by upregulating PD-L1.
Experimental procedures. After the optimization process, we exposed 50 established melanoma cell lines to interferons (alpha, beta and gamma) and measured PD-L1, pSTAT1 (Y701), pSTAT3, pSTAT5, pSTAT6, NF-kB, pERK (1/2), pAKT, pP38, cleaved PARP, CyD3 expression level by flow cytometry (FACS LSRII) and PD-L1 as well as STAT1 transcript level was checked by quantitative real time PCR. Western blots were performed to further analyze interferon receptor signaling in selected cells lines.
Results. Of 50 cell lines tested, 44 responded to at least one interferon ranging from 2 to 10-fold increase in PD-L1 compared to baseline expression, and 5 cell lines failed to respond to interferons with PD-L1 upregulation. Measurement of PD-L1 transcript level by qPCR as well as surface expression level by flow cytometry demonstrated that PD-L1 transcript level was up-regulated close to 20 fold by IFNγ in 30 minutes for two representatives of good PD-L1 responders. Phosphoflow experiments confirmed that 3 representative good responding cell lines had significant up-regulation of pSTAT1 compared to the baseline. However, among non-PD-L1 responding cell lines M412B showed significant change in pSTAT1, but WM1366 and M395 did not both, by phosphoflow and western blot analyses. M368 is a cell line that did not up-regulate PD-L1 upon exposure to interferon gamma, but it did up-regulate PD-L1 when exposed to type I interferons (alpha and beta). This cell line showed loss of JAK2 expression. WM1366 showed low JAK2 expression with loss of pJAK2 and SOCS1 overexpression, which is consistent with non-significant up-regulation of PD-L1 upon interferon treatment.
Conclusions. PD-L1 is inducible in the majority of melanoma cell lines tested, but approximately 10% of them are not able to up-regulate PD-L1 expression upon interferon exposure. JAK-STAT pathway is critically important in regulation of inducible expression of PD-L1, and we have defined molecular defects in individual cell lines, which did not up-regulate PD-L1 expression. This information may be important to assess clinical data from PD-1/L1 blockade trials as to whether intact interferon receptor pathway inducible response may better predict the response of anti-PD-1/L1 immunotherapy than baseline PD-L1 levels.
Citation Format: Daniel S. Shin, Angel Garcia-Diaz, Helena Escuin-Ordinas, Nicolaos J. Palaskas, Sara M. Komenan, Thomas G. Graeber, Begonya Comin-Anduix, Antoni Ribas. Toward better selection of PD-1 blockade responders through direct markers of adaptive immune resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5013. doi:10.1158/1538-7445.AM2015-5013