Peripheral biomarkers associated with response and resistance to anti-PD-1 therapy in metastatic melanoma patients represent an unmet medical need. High neutrophil activity is associated with immune checkpoint inhibitor failure, amongst others due to release of neutrophil extracellular traps (NETs) that have been shown to protect tumor cells against cytotoxic attacks. Human neutrophil elastase (HNE) and calprotectin are the major NETs constituents. We hypothesized that HNE mediated degradation of calprotectin (CPa9-HNE), when measured in serum, has peripheral biomarker potential in metastatic melanoma patients treated with anti-PD-1 therapy.


A monoclonal antibody was raised against a specific HNE mediated degradation fragment of calprotectin (CPa9-HNE) and used to develop an ELISA for quantifying CPa9-HNE in serum. CPa9-HNE was then measured in pre-treatment serum from metastatic melanoma patients treated with anti-PD-1 therapy (pembrolizumab) (n=35). The biomarker levels were associated with progression-free survival (PFS) and overall survival (OS) by univariate and multivariate Cox regression analyses adjusting for PDL1 expression (≥1%), lactate dehydrogenase (LDH), BRAF mutational status, and c-reactive protein (CRP).


High pre-treatment CPa9-HNE levels predicted worse PFS (HR=3.32, 95%CI=1.25-8.82, p=0.016) and OS (HR=11.31, 95%CI=2.27-56.33, p=0.003) when compared to low CPa9-HNE levels by univariate Cox regression. By multivariate Cox regression, high CPa9-HNE was found to be independently predictive of poor PFS (HR=8.22, 95%CI=1.30-52.14, p=0.025) and OS (HR=76.87, 95%CI=4.73-1248.57, p=0.002) when adjusted for PDL1 expression, LDH, BRAF mutations, and CRP.


A non-invasive biomarker of HNE mediated degradation of calprotectin (CPa9-HNE) was associated with poor PFS and OS in metastatic melanoma patients treated with anti-PD-1 therapy. These findings support a link between neutrophil activity/NETs and response to anti-PD-1 and indicate promising non-invasive biomarker potential to stratify melanoma patients for anti-PD-1 therapy in future clinical studies.

Citation Format: Christina Jensen, Anders Kverneland, Marco Donia, Joachim H. Mortensen, Morten A. Karsdal, Inge Marie Svane, Nicholas Willumsen. Evaluation of CPa9-HNE - a peripheral biomarker based on neutrophil extracellular traps (NETs) - for predicting outcome in patients with metastatic melanoma treated with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 386.