Proteomic profiling of a patient with cutaneous melanoma metastasis regression following topical contact sensitizer diphencyprone and immune checkpoint inhibitor treatment

Variations in melanoma metastasis clinical responses during the dual therapy treatment course

At baseline, the patient presented with extensive in-transit metastases of the right lower extremity (Fig. 1a). Following DPCP topical monotherapy, a diffuse delayed-type hypersensitivity reaction occurred with mixed tumor regression response (Fig. 1b). Although some lesions appear larger, a few lesions appear smaller. Infiltration of immune cells may lead to the appearance of increased tumor size, a phenomenon known as pseudo-progression⁹. Following pembrolizumab monotherapy, there was severe worsening and progression of disease involvement (Fig. 1c). During the dual treatment period, there was substantial regression of melanoma metastases (Fig. 1d).

DPCP and pembrolizumab have contrasting effects on immune checkpoint protein levels in tissue when used as monotherapy

Within the tissue samples, protein levels of checkpoint inhibitor proteins PD-1, PD-L1, and lymphocyte-activation gene 3 (LAG-3) rose from baseline until the discontinuation of DPCP monotherapy at Day 80 by 22.7%, 36.9%, and 56.4%, respectively. Pembrolizumab monotherapy was initiated at Day 80. PD-1, PD-L1, and LAG-3 decreased by 19.9%, 11.5%, and 6.7%, respectively, between Day 80 and Day 202 (Figs. 1e–g and 2a).

Dual therapy with pembrolizumab and DPCP is associated with increased expression of various proteins

Dual treatment with pembrolizumab and DPCP was associated with rebound increases of PD-1, PD-L1, and LAG-3 (24.7%, 21.3%, and 44.6%, respectively) between Day 202 and Day 385 (Fig. 1e–g) . Serum protein levels for immune checkpoint proteins were not available at the onset of dual therapy, but generally trended upwards during all periods (Figs. 1e–g and 2b). Proteins associated with tumor regression including granzyme A (GZMA) and Th1 markers such as the CXC motif chemokine ligand family (CXCL9, CXCL10, CXCL11) and interferon gamma (IFNG) mirrored the checkpoint inhibitor decreases seen in tissue during single agent pembrolizumab therapy. These proteins then rebounded following initiation of dual therapy with DPCP. Tissue protein levels of tumor necrosis factor superfamily member 14 (TNFSF14), a protein with antitumor activity¹⁰, decreased during pembrolizumab monotherapy by 35.5%, but levels rebounded by 41.2% upon dual therapy initiation. Markers of inflammation and neutrophil chemotaxis¹¹ including CXCL1, interleukin (IL)-6, and IL-8 also followed this pattern with a high degree of induction during dual therapy (283.9%, 78.6%, and 215.5%, respectively). Additional markers of inflammation and monocyte chemotaxis including monocyte-chemoattractant protein-1 (MCP-1) and MCP-2 as well as tissue remodeling marker matrix metallopeptidase 12 (MMP12) followed a similar pattern (Table 1 and Fig. 3).

Tissue protein expression of proteins discussed in Table 1, plotted throughout the treatment course. Proteins are color coded by pathway and function. Five groups are represented including immune checkpoint proteins (PD-1, PD-L1, and LAG-3), tumor regression proteins (TNFSF14, GZMA), tumor promoting proteins (CCL17, CD70, LAMP3), inflammatory proteins/chemotactants (CXCL1, IL-6, IL-8, MCP-1, MCP-2, MMP12), and Th1 markers (CXCL9, CXCL10, CXCL11, IFNG).

Dual therapy with pembrolizumab and DPCP suppresses markers of tumor progression to a higher degree than monotherapy with either agent

C–C Motif Chemokine Ligand-17 (CCL17) is a tumor promoting protein that activates regulatory T cells thereby inhibiting the antitumor response. Levels of CCL17 in tissue samples rose by 47.8% from baseline to Day 80 during DPCP monotherapy and further increased by 74.3% during pembrolizumab monotherapy. In contrast, a 10.3% decrease was observed during dual therapy. Cluster of differentiation-70 (CD70) is a protein implicated in tumor cell survival that also activates regulatory T cells and skews T cells toward exhaustion. Protein levels of CD70 in tissue samples decreased from baseline to Day 80, further from Day 80 to Day 202, and by the highest score between Day 202 and Day 385 (2.9%, 17.7%, and 30.0%, respectively), as did serum levels from DPCP monotherapy (2.7%) to dual therapy (6.9%). Lysosome associated membrane protein-3 (LAMP3) represents another tumor promoting protein for which the greatest downregulation was observed during dual therapy (35.0%) compared to DPCP monotherapy (16.5%) or pembrolizumab monotherapy (2.1%). Serum levels of LAMP3 mirrored these decreases with downregulation during DPCP monotherapy (7.9%) and further reduction during dual therapy (15.1%) (Table 1 and Fig. 3).