Abstract
Abstract Background: Blockade of the immunosuppressive PD-1/PD-L1 pathway has shown promising clinical results in patients with treatment-refractory solid tumors including melanoma. PD-L1, a protein displayed by many tumors, ligates the PD-1 co-inhibitory receptor on activated T cells. We previously found that IFN-γ, a potent inducer of PD-L1, was expressed by TILs in PD-L1(+) but not PD-L1(-) melanomas, creating an immunosuppressive microenvironment by a mechanism that we term “adaptive immune resistance” (Taube et al., Science Transl Med 2012). In the current study, we assessed other factors associated with PD-L1 expression in melanoma through differential gene expression analysis, in order to better understand the biology of this pathway and identify potential targets for combination immunotherapies. Methods: PD-L1(+) vs. (-) melanomas including immune cell infiltrates (n=11) were laser-capture microdissected (LCM) from formalin-fixed paraffin embedded (FFPE) specimens and analyzed by whole genome array analysis with cDNA-mediated Annealing, Selection, extension and Ligation (DASL), a novel technology that allows for gene expression analysis with partially degraded mRNAs obtained from FFPE specimens. Differentially expressed genes were submitted to the NIH functional analysis tool DAVID to search for enrichment in biologically related groups. Multiplex quantitative (q)RT-PCR was used to validate differential expression of genes of interest and to examine other candidate genes in new set of PD-L1(+) vs.(-) melanomas (n=11). Results: DASL/DAVID analysis of PD-L1(+) vs. PD-L1(-) melanomas uncovered genes of interest in several immunologically relevant pathways, including “Defense Response” (p<1.5E-9) and “T-cell activation” (p<2.2E-5). Multiplex qRT-PCR to examine genes of interest from the DAVID analysis along with other potentially relevant candidate genes was normalized to the GUSB housekeeping gene or to CD45 (pan-leukocyte marker), revealing over-expression of genes associated with immunosuppression (PD-1, PD-L1, LAG-3, IL-10), activated CD8 T cells (CD8A, IFN-γ, lysozyme, perforin, CCL5/RANTES), and antigen presenting cells (CD163, TLR3, CXCL1) in PD-L1(+) melanomas (p<0.10). Conclusions: These experiments identified groups of functionally related, differentially expressed immuno-regulatory genes in PD-L1(+) melanomas. These factors may coordinately create an immunosuppressive tumor microenvironment, by synergizing to enhance PD-L1 expression on tumor cells and to inhibit T cell function. Immunohistochemical and in vitro functional validation assays of candidate gene products are currently underway in order to identify new ways to overcome adaptive immune resistance in synergistic treatment combinations with PD-1/PD-L1 blockade. Citation Format: Geoffrey D. Young, Tracee L. McMiller, Haiying Xu, Shuming Chen, Alan E. Berger, Jinshui Fan, Robert A. Anders, Christopher Cheadle, Drew M. Pardoll, Suzanne L. Topalian, Janis M. Taube. Differential expression of immuno-regulatory genes associated with PD-L1 display: Implications for clinical blockade of the PD-1/PD-L1 pathway in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2013-446
Published Version
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