Over the last decade, immune checkpoint inhibitors (ICIs) have improved the overall survival (OS) of patients with metastatic cancer. Monoclonal antibodies directed to the programmed cell death protein 1 (PD-1) receptor have emerged as frontline therapies for cancers such as metastatic melanoma. However, there is an unmet need for biomarkers to guide treatment decisions, as the efficacy of ICIs is highly variable across individual patients. Although the expression of its ligand programmed death-ligand 1 (PD-L1) has been positively associated with clinical outcome in metastatic melanoma, the predictive performance remains limited, which may partly be explained by tumor heterogeneity.1 Similarly, other biomarkers, such as CD8⁺ T cell infiltration and tumor mutational burden, individually or combined, have been considered as biomarkers.2 3 Interestingly, recent evidence suggests that transforming growth factor beta (TGF-β), cancer-associated fibroblasts (CAFs) and immune-exclusion in tumor tissue are associated with worse clinical outcome after PD-L1 or PD-1 inhibition in solid cancer.4–6 Those immune-excluded tumors are characterized by a collagen-rich peritumoral stroma (tumor fibrosis) that blocks the interaction between CD8⁺ T cells and tumor cells.4–6 The main collagens of the interstitial matrix (type I and III collagen) and the basement membrane (type IV collagen) are upregulated in immune-excluded tumors that are accompanied by a peritumoral location of CD8⁺ T cells.7 Moreover, components of proteolytic extracellular matrix (ECM) turnover appear to affect the response mechanisms to ICIs by regulating steps in the cancer-immunity cycle.8 Melanoma is characterized by a highly reactive and fibrotic tumor stroma.9 10 Increased stroma activity leads to increased matrix metalloproteinase (MMP)-mediated collagen degradation resulting in the generation and release of small protein fragments containing specific protease-generated neo-epitopes into the blood circulation.11 12 These ECM protein fragments can be used as non-invasive biomarkers measured in blood, and may be used to assess processes such as MMP-mediated collagen degradation and collagen formation and cross-linking by evaluating its blood-based signature.12 This has been demonstrated by our previous study showing that biomarkers measuring N-terminal pro-peptide of type III collagen (PRO-C3) or MMP-degraded type IV collagen (C4M) are associated with worse clinical outcome after cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs in patients with metastatic melanoma.13 The two collagen-derived biomarkers assess different components of the ECM in the tumor microenvironment (TME) by reflecting a dense stromal interstitial matrix and fibrotic activity (PRO-C3) and degradation of the basement membrane (C4M).14

The current study aimed to investigate the relationship of tissue-derived biomarkers and clinical outcome after PD-1 inhibition in patients with metastatic melanoma. Protein fragments reflecting type III collagen formation (PRO-C3) and cross-linking (PC3X), MMP-degraded type III (C3M) and IV collagen (C4M), granzyme B-degraded type IV collagen (C4G) and citrullinated and MMP-degraded vimentin (VICM) were measured in serum obtained before and 6 weeks after the first administration of PD-1 ICIs. Moreover, the dynamics of collagen turnover and VICM during treatment were assessed.

Serum from a total of 107 patients with metastatic melanoma at baseline and paired serum from 94 patients (88% of all patients) that was obtained 6 weeks (median 6, IQR 6–9 weeks) after PD-1 ICI therapy was available for analysis. The baseline patient characteristics including demographic and clinical data are shown in table 1. Patients generally received PD-1 ICIs as first-line treatment shortly after diagnosis (82% of all patients), and therefore generally have a good performance status (WHO PS 0 or 1: 92% of all patients). All 107 patients were evaluable for the OS analysis, with a median OS of 36.2 months. Three patients (3%) were not evaluable for determination of BOR, that is, because of early clinical progression, resulting in a disease control rate (the proportion of patients with a BOR of SD, PR or CR) of 63.5%. The serum levels of the six different ECM biomarkers were evaluated at baseline: PRO-C3, PC3X, C4G, C3M, C4M and VICM. The distribution of those baseline measurements is shown in online supplemental figure 1. For all six ECM biomarkers, there was a right-skewed distribution of data.

The present study demonstrates that blood-based biomarkers of protease-generated neo-epitopes on protein fragments reflecting altered ECM turnover are associated with clinical outcome after PD-1 ICI therapy in metastatic melanoma. Baseline serum levels of ECM protein fragments PRO-C3 and PC3X, indicating a collagen-rich peritumoral stroma and fibrotic activity, were associated with resistance to PD-1 inhibition. Moreover, upregulation of VICM during therapy, reflecting M1 macrophage activity, was associated with improved survival.

Patients with higher PRO-C3 and PC3X levels had worse clinical outcome after PD-1 inhibition compared with patients with lower levels. Interestingly, as PRO-C3 and PC3X are released to the blood circulation during excessive collagen formation and cross-linking,16 the findings suggest that those markers identify tumors with an immune-excluded phenotype. Immune-exclusion is characterized by dense collagen stroma that limits the entry of tumor infiltrating lymphocytes into the TME, which is considered as an important biological mechanism of therapy resistance.4 17 Cytotoxic T cell activity towards cancer cells in the TME is suggested to be an independent factor of the PD-L1 tumor proportion score and tumor mutation burden.2 18

Although these protease-generated protein fragments were significantly correlated to LDH levels, we demonstrated that higher PRO-C3, independent of LDH, was associated with worse PFS and OS and that higher PC3X was associated with worse PFS. LDH is known as a marker of tumor burden and a clinically significant adverse prognostic factor for metastatic melanoma, which is also confirmed in this immuno-oncology setting.19 Elevated LDH levels is integrated as a suffix to the M1 subcategories in recent TNM cancer staging protocols,20 providing additional prognostic value. Interestingly, similar observations for PRO-C3 were previously seen in patients with metastatic melanoma who were treated with CTLA-4 ICI therapy,13 suggesting that the non-invasive biomarker may be considered across different ICIs.

Dense collagen stroma may also affect the diffusion and distribution of the therapeutic antibodies,21 in addition to its effect in limiting the entry of tumor infiltrating lymphocytes into the TME. Previous work by Jensen et al 13 demonstrated additional prognostic value of the ratio of type III collagen degradation to formation (C3M/PRO-C3) after CTLA-4 inhibition in metastatic melanoma. The present findings confirm that patients with a high C3M/PRO-C3 ratio (>1) are linked to better clinical outcome after PD-1 inhibition, thereby providing additional evidence for the hypothesis that patients with relatively low net fibrosis/collagen deposition are more likely to respond to ICIs. PRO-C3 and PC3X are associated with a higher activity of CAFs. In addition to the desmoplastic, collagen-rich TME related to immune-exclusion, CAFs have also shown to contribute to a diminished immune function by expressing TGF-β, PD-L1/PD-L2 and by inducing immune checkpoints on T cells.6 22–24

We demonstrated that patients with an increase in VICM during treatment had improved clinical outcome. VICM is released from activated inflammatory M1 macrophages,25 which may be an important factor for response to ICIs compared with the immune-suppressive M2 macrophage phenotype. For example, PD-L1 inhibition was demonstrated to polarize macrophages into a more pro-inflammatory phenotype, which was associated with enhanced T cell activity.26 In mice, PD-1 inhibition was found to increase macrophage phagocytosis, and was associated with better survival in a macrophage-dependent fashion.27 Our results support these findings and additionally suggest that blood-based VICM levels have potential value for monitoring early response to PD-1 inhibition.

Whereas higher C4M levels at baseline were previously demonstrated to be associated with worse clinical outcome after CTLA-4 inhibition in metastatic melanoma,13 we could not confirm this observation after PD-1 inhibition. C4M mirrors basement membrane remodeling, which is strongly associated with malignant progression of cancer.28 These inconsistent results may be explained either by the difference of mechanism of action of CTLA-4 and PD-1 inhibition or by the higher efficacy of PD-1 inhibition in this immuno-oncology setting.13

Interestingly, C4G levels were previously associated with improved OS after CTLA-4 inhibition in metastatic melanoma.29 This C4G biomarker measures a neo-epitope of C4G that may reflect cytotoxic T-cell infiltration in the TME.30 Nevertheless, we could not observe a relationship between C4G levels and clinical outcome after PD-1. Granzyme B is a key serine protease that is secreted by activated T and NK cells to induce apoptosis.31 While it is suggested to contribute to response mechanisms of PD-1 inhibition in metastatic lung cancer, illustrated by an inverse relationship of both granzyme B levels as well as germline variation of GZMB and clinical outcome,32 further research is needed to elaborate on the differences between ICIs and tumor types.33

Despite the interesting findings, this study also has some limitations. Patients were treated with two different ICIs (nivolumab or pembrolizumab) and not designed/powered to differentiate between the two. Although these are highly similar monoclonal antibodies directed to the PD-1 receptor, this may have an impact on the results. Furthermore, the sample size was too small to correct for multiple testing or for splitting the patients into a discovery and validation group. Nevertheless, in the current analysis we were able to demonstrate strong associations with survival outcomes for a selection of ECM biomarkers. Although ECM turnover is a physiological process taking place throughout the body, the assessments of specific post-translational modifications (neo-epitopes) may reduce the systemic background of healthy turnover and instead increase the specificity of ECM turnover in the TME.12 34 Besides, even though our main objective was to investigate the association of markers with clinical outcome, the findings do not address the predictive performance of the biomarkers, for which prospective external validation is needed in cohorts randomized to different modalities. Lastly, a statistical methodology is needed to determine the cut-off discriminating high and low levels of these molecules. This is of particular importance for validation in a randomized setting for establishing the performance of biomarkers for diagnostic use, which was not the scope of this study. Here, cutoffs may be selected by receiver operating characteristics. We believe that identification of patients who have primary resistance should require a high negative predictive value, consequently with stringent cut-off values.

In conclusion, we demonstrated that the ECM protein fragments PRO-C3 and PC3X in baseline serum, representing a dense collagen stroma, were associated with worse clinical outcome after PD-1 inhibition in metastatic melanoma. Moreover, increasing VICM levels early during therapy was associated with improved survival. VICM levels correspond with M1 macrophage activity by its specific release of MMP-degraded and citrullinated vimentin in the blood circulation. After validation, these biomarkers may provide a robust non-invasive tool for patient stratification and therapeutic decision-making in immuno-oncology.