Furthermore, several of these proteins remained consistently associated with PFS after adjusting for age, sex, and abnormal LDH levels in sensitivity multivariate analyses, for example, PRAP1, DSC3, C1QC, LAMA2, CCL2, CCL3, CCL4, IL-6, and VEGFA. The PFS is a reliable treatment outcome that is directly linked to the Ro 28-1675 treatment effect and less affected by subsequent treatment confounders that can affect OS. 4. Abstract Background Immune checkpoint inhibitors (ICIs) have significantly improved the outcome in metastatic cutaneous melanoma (CM). However, therapy response is limited to subgroups of patients and clinically useful predictive biomarkers are lacking. Methods To discover treatment-related systemic changes in plasma and potential biomarkers associated with treatment end result, we analyzed serial plasma samples from 24 patients with metastatic CM, collected before and during ICI treatment, with mass-spectrometry-based global proteomics (high-resolution isoelectric focusing liquid chromatographyCmass spectrometry (HiRIEF LC-MS/MS)) and targeted proteomics with proximity extension assays (PEAs). In addition, we analyzed plasma proteomes of 24 patients with metastatic CM treated with mitogen-activated protein kinase inhibitors (MAPKis), to pinpoint changes in protein plasma levels specific to the ICI treatment. To detect plasma proteins associated with treatment response, we performed stratified analyses in anti-programmed cell death protein 1 (anti-PD-1) responders and non-responders. In addition, we analyzed the association between protein plasma levels and progression-free survival (PFS) by Cox proportional hazards models. Results Unbiased HiRIEF LC-MS/MS-based proteomics showed plasma levels alterations related to anti-PD-1 treatment in 80 out of 1160 quantified proteins. Circulating PD-1 experienced the highest increase during anti-PD-1 treatment (log2-FC=2.03, p=0.0008) and in anti-PD-1 responders (log2-FC=2.09, p=0.005), but did not change in the MAPKis cohort. Targeted, antibody-based proteomics by PEA confirmed this observation. Anti-PD-1 responders experienced an increase in plasma proteins involved in T-cell response, neutrophil degranulation, inflammation, cell adhesion, and immune suppression. Furthermore, we discovered new associations between plasma proteins (eg, interleukin 6, interleukin 10, proline-rich acidic protein 1, desmocollin Ro 28-1675 3, C-C motif chemokine ligands 2, 3 and 4, vascular endothelial growth factor A) and PFS, which may serve as predictive biomarkers. Conclusions We detected an increase in circulating PD-1 during anti-PD-1 treatment, as well as diverse immune plasma proteomic signatures in anti-PD-1 responders. This study demonstrates the potential of plasma proteomics as a liquid biopsy method and in discovery of putative predictive biomarkers for anti-PD-1 treatment in metastatic CM. observed that even though immune system responds with a PD-1+ CD8+ T-cell infiltration and an inflammatory response after a single dose of anti-PD-1 ICIs, the tumor develops resistance mechanisms of immune suppression and tumor development in response to treatment.39 Furthermore, it is likely that this role of these molecules is complex and depending on the cell environment, as it is the case for IL-10, an established immunosuppressive protein that has been demonstrated to induce a strong antitumor T-cell response in mice and humans.43 44 Several of the proteins that were differentially altered (-up/-down) in plasma of anti-PD-1-R, as compared with anti-PD-1-NR were also predictive of PFS. Furthermore, several of these proteins remained consistently associated with PFS after adjusting for age, sex, and abnormal LDH levels in sensitivity multivariate analyses, for example, PRAP1, DSC3, C1QC, PSFL LAMA2, CCL2, CCL3, CCL4, IL-6, and VEGFA. The PFS is usually a reliable treatment end result that is directly linked to the treatment effect and less affected by subsequent treatment confounders that can affect OS. Analyzing the association with PFS can show the role of the plasma proteins as potential biomarkers and the biological processes in which they are involved, which favor or hinder response to treatment. Curiously, in the PFS survival analyses high pre-trm levels of a Ro 28-1675 subset of inflammatory proteins were associated with shorter PFS for both the ICI and MAPKi cohort, whereas an increase in their levels during ICIs treatment was associated with a protective effect and longer PFS (ie, IL-6, CCL2, CCL3, CCL4, and VEGFA). This emphasizes the importance of timing in plasma sampling and how the temporal effects affect the role of proteins as biomarkers. Last, in a proof-of-concept analysis, we also show that by employing a proteogenomics approach we can detect proteins harboring coding variants, similar to the liquid biopsy methods to detect cell free DNA, an approach Ro 28-1675 that has shown to reflect the overall mutational profile of tumors as accurately as singe biopsies.12 Conclusions In this discovery study, we demonstrated increased levels of circulating PD-1 and PD-L1 in plasma of patients with metastatic CM during anti-PD-1 treatment, as well as diverse immune plasma proteomic signatures, which require validation in indie larger cohorts with targeted methods. Moreover, we spotlight the potential of combined, global,.