Defining plasma proteomic biomarkers features of response and resistance to immune checkpoint blockade in melanoma

Most patients treated with immune checkpoint blockade (ICB) do not have durable treatment responses, stressing a critical need to identify early non-invasive biomarkers of response. Circulating biomarkers provide easy access for serial monitoring and can provide insight on the mechanisms of response to ICB. We performed plasma proteomics (~3000 proteins) from 250 metastatic melanoma patients before and on ICB treatment and performed analysis to identify response- and time point-associated peripheral protein biomarkers. We next generated patient-matched peripheral blood lymphocyte (PBL) surface proteomic data to measure their proportions in circulation. We further obtained 13 patient-matched tumor samples and performed single-cell RNA-sequencing (scRNA-seq). We fit linear mixed effects (LME) models to predict plasma protein abundances, using the sample time point, response status and interaction term between time point and response as covariates, with patient ID as a random effect to account for patient-level differences in protein abundance. We identified 397 proteins with a significant time point, response, or interaction effect, or some combination of these effects. For proteins with positive time point effects (upregulated post-ICB), gene ontology analysis suggested these proteins were associated with inflammatory pathways involving the activation of effector immune functions. LME models fitted on the PBL proportions data and an analysis of immune-related plasma proteins supported that post-ICB samples may be defined by increased immune activity, including T cell infiltration. Moreover, expression of genes corresponding to these post-ICB proteins in the tumor scRNA-seq data tended to be favored by immune subsets, namely those involved in immune cell recruitment and tumor reactivity, such as CXCL10+ macrophages and CXCL13+ T cells, respectively. On the other hand, expression genes corresponding to plasma proteins upregulated in non-responders in the TME tended to be favored by suppressive myeloid subsets such as SPP1+ macrophages as well as malignant cells, suggesting the potential of immunosuppressive and pro-tumor biological processes to be involved in ICB resistance. Inference of cell-cell interactions in the TME further showed the involvement of these non-responder genes in potentially immunosuppressive and pro-tumor processes. Effforts to examine absolute levels of key secreted proteins using the T48 panel are ongoing. Together, these data represent one of the deepest peripheral biomarker studies using paired samples in melanoma patients treated with anti-PD1 therapy.