Abstract 3166: The degree of conserved interferon gamma signaling guides response to immune checkpoint blockade therapy
Authors: Grasso Catherine , Tsoi Jennifer , Onyshchenko Mykola , Abril-Rodriguez Gabriel , Champhekar Ameya , Medina Egmidio , Torrejon Davis , Shin Daniel , Quist Michael , Ross-Mcdonald Petra , Wind-Rotolo Megan , Speiser Daniel , Kalbasi Anusha , Ribas Anthony 
Published: September 2020

Abstract
The long-lasting responses to immune blockade therapy in a subset of patients with melanoma remain largely unexplained. We assessed baseline and on-therapy biopsies from 118 patients with advanced melanoma treated with the anti-PD-1 antibody nivolumab alone (N = 82) or in combination with the anti-CTLA-4 antibody ipilimumab (N = 36) (CheckMate 038 study). There were 46 progressive disease (PD) cases, 26 stable disease (SD) cases, and 46 complete or partial response (CRPR) cases. Tumors were analyzed by RNA sequencing. RNAseq bulk immune cell deconvolution revealed that the largest immune cell change on-treatment was an increase in CD8+ T cell infiltration for patients regardless of treatment or clinical response (P &lt; 0.04, 0.02, 2e-4 for PD, SD, CRPR). In CRPR, there was a corresponding increase in CD4+ T cells (P &lt; 2e-4), Tregs (P &lt; 9e-4), NK cells (P &lt; 3e-5) and macrophages (P &lt; 3e-5). Principal component analysis showed that T cell infiltration was the best organizer of the RNAseq biopsies; 3694 genes were correlated or negatively correlated with T cell infiltration.
Analysis of 58 human melanoma cell lines exposed in vitro to interferon-gamma (IFNγ) for 6 hours showed a conserved response to this cytokine in all cell lines able to signal through the IFNγ receptor (N = 46), including an increase in antigen presentation machinery, an increase in expression of IFNγ signaling molecules representing a signal amplification, and production of chemokines that attract other immune cells, while there was a decrease in cell cycle and WNT signaling pathways. These same gene sets are present in the list of genes correlated and negatively correlated with T cell infiltration in the patient data, as well as the list of genes significantly up- or down-regulated in CRPR cases. Only cell lines with JAK1 or JAK2 mutations (N = 12) did not exhibit this conserved response; in fact, these mutants had no genes with at least a 2-fold change.
Analysis of the patient biopsies using the IFNγ regulated gene sets revealed that in CRPR cases there was a large increase in HLA expression (P &lt; 0.04, 4e-3, 8e-4 for HLA-A, HLA-B, HLA-C), along with a corresponding large decrease in WNT signaling (P &lt; 3e-4), a driver of immune exclusion. We did not find genomic alterations (in the subset of cases that we exome sequenced) or gene expression differences pre-treatment to explain the greater response in the CRPR cases, including no JAK1/2 mutations. The lack of naturally occurring mutations disabling IFNγ signaling in our baseline biopsies is likely selected for by the role IFNγ plays in up-regulating PD-L1 expression necessary for establishing the anti-PD1 immune blockade. Using RNAseq immune cell deconvolution, the 36 CRPR cases (16 monotherapy, 20 combination) had significantly larger CD8+ T cell infiltration pre-treatment (P &lt; 8e-4 compared to PD cases). Therefore, our data showed that the strength of the antitumor T cell response and corresponding downstream IFNγ signaling is the main driver of response or resistance to immune checkpoint blockade therapy.
Citation Format: Catherine Grasso, Jennifer Tsoi, Mykola Onyshchenko, Gabriel Abril-Rodriguez, Ameya Champhekar, Egmidio Medina, Davis Torrejon, Daniel Shin, Michael Quist, Petra Ross-Mcdonald, Megan Wind-Rotolo, Daniel Speiser, Anusha Kalbasi, Anthony Ribas. The degree of conserved interferon gamma signaling guides response to immune checkpoint blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3166.