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Immune-checkpoint inhibitors (ICIs) have revolutionized melanoma treatment, yet approximately half of patients do not respond to these therapies. Identifying prognostic biomarkers is crucial for treatment decisions. Our retrospective study assessed liquid biopsies and tumor tissue analyses for two potential biomarkers: danger-associated molecular pattern (DAMP) S100A8/A9 and its source, neutrophils. In 43 metastatic unresected stage III/IV melanoma patients, elevated serum levels of S100A8/A9 and neutrophils before and during ICI treatment correlated with worse outcomes. Furthermore, in 113 melanoma patients, neutrophil expression in the tumor microenvironment (TME) was associated with relapse and reduced survival. Measuring S100A8/A9 and neutrophils could enhance immunotherapy monitoring by predicting impaired clinical outcomes and non-response to ICIs. Serum S100A8/A9 levels and neutrophil counts at baseline (T0) and during treatment (T3) correlated with reduced progression-free survival (PFS). Elevated S100A8/A9 levels at T0 and T3 negatively impacted overall survival (OS). Notably, neutrophil infiltration was more prevalent in primary melanomas than in nevi and metastases, and its presence in primary melanomas was linked to poorer survival. S100A8/A9 serum levels, neutrophil counts, and tumor-associated neutrophil infiltration represent promising biomarkers for predicting treatment response and clinical outcomes in melanoma patients receiving ICIs. Significance: These findings underscore the critical need for reliable biomarkers in melanoma research, particularly for predicting responses to immune-checkpoint inhibitors (ICIs). Identifying S100A8/A9 levels and neutrophil infiltration as potential indicators of treatment outcomes offers valuable insights for personalized therapy decisions. By enhancing monitoring and prognosis assessment, these biomarkers contribute to refining treatment strategies, ultimately improving patient care and outcomes. This research bridges gaps in understanding melanoma response mechanisms and highlights avenues for further investigation into immune-related markers, fostering advancements in precision medicine for melanoma patients.

Abstract Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Background An increased incidence of thrombotic complications associated with an increased mortality rate has been observed under immune checkpoint inhibition (ICI). Recent investigations on the coagulation pathways have highlighted the direct role of key coagulatory proteins and platelets in cancer initiation, angiogenesis and progression. The aim of this study was to evaluate the prognostic value of von Willebrand factor (vWF) and its regulatory enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), D-dimers and platelets in a cohort of patients with metastatic melanoma receiving ICI.Methods In a prospective cohort of 83 patients with metastatic melanoma, we measured the systemic levels of vWF-antigen (vWF:Ag), ADAMTS13 activity, D-dimers and platelets, before the beginning of the treatment (baseline), and 6, 12 and 24 weeks after. In parallel, we collected standard biological parameters used in clinical routine to monitor melanoma response (lactate deshydrogenase (LDH), S100). The impact of neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) on overall survival (OS) in patients receiving ICI was assessed. Univariable and multivariable Cox proportional models were then used to investigate any potential association of these parameters to clinical progression (progression-free survival (PFS) and OS). Baseline values and variations over therapy course were compared between primary responders and resistant patients.Results Patients with melanoma present with dysregulated levels of vWF:Ag, ADAMTS13 activity, D-dimers, LDH, S100 and CRP at the beginning of treatment. With a median clinical follow-up of 26 months, vWF:Ag interrogated as a continuous variable was significantly associated with PFS in univariate and multivariate analysis (HR=1.04; p=0.007). Lower values of vWF:Ag at baseline were observed in the primary responders group (median: 29.4 µg/mL vs 32.9 µg/mL; p=0.048) when compared with primary resistant patients. As for OS, we found an association with D-dimers and ADAMTS13 activity in univariate analysis and vWF:Ag in univariate and multivariate analysis including v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and Eastern Cooperative Oncology Group (ECOG) performance status. Follow-up over the course of treatment depicts different evolution profiles for vWF:Ag between the primary response and resistance groups.Conclusions In this prospective cohort, coagulatory parameters such as ADAMTS13 activity and D-dimers are associated with OS but baseline vWF:Ag levels appeared as the only parameter associated with response and OS to ICI. This highlights a potential role of vWF as a biomarker to monitor ICI response of patients with malignant melanoma.

The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell‐free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell‐free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (next‐generation sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high‐quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained. However, the choice of blood collection tubes is a critical pre‐analytical variable.