Regression of melanoma metastases after immunotherapy is associated with activation of antigen presentation and interferon-mediated rejection genes

Abstract

We present the results of a comparative gene expression analysis of 15 metastases (10 regressing and 5 progressing) obtained from 2 melanoma patients with mixed response following different forms of immunotherapy. Whole genome transcriptional analysis clearly indicate that regression of melanoma metastases is due to an acute immune rejection mediated by the upregulation of genes involved in antigen presentation and interferon mediated response (STAT-1/IRF-1) in all the regressing metastases from both patients. In contrast, progressing metastases showed low transcription levels of genes involved in these pathways. Histological analysis showed T cells and HLA-DR positive infiltrating cells in the regressing but not in the progressing metastases. Quantitative expression analysis of HLA-A,B and C genes on microdisected tumoral regions indicate higher HLA expression in regressing than in progressing metastases. The molecular signature obtained in melanoma rejection appeared to be similar to that observed in other forms of immune-mediated tissue-specific rejection such as allograft, pathogen clearance, graft versus host or autoimmune disease, supporting the immunological constant of rejection. We favor the idea that the major factor determining the success or failure of immunotherapy is the nature of HLA Class I alterations in tumor cells and not the type of immunotherapy used. If the molecular alteration is reversible by the immunotherapy, the HLA expression will be upregulated and the lesion will be recognized and rejected. In contrast, if the defect is structural the MHC Class I expression will remain unchanged and the lesion will progress.

Figure 1

expression pattern clustering of melanoma metastases obtained from comparing regressing versus progressing metastases (p = 0.001). Genes upregulated in regressing metastases are represented in red, genes overexpressed in progressing metastases are represented in green. Most of the genes upregulated in regressing metastases belong to the immune response. Key genes involved in immunological rejection (antigen presentation, immune effectors factors and interferon stimulates genes) are included in the orange box, metabolism genes upregulated in progressing metastases are included in the green box.

Figure 2

Class comparison between progressing and regressing metastases in both patients. (a) Top 15 first canonical pathways ranking according to significance level (Fisher exact test −log p-value) based on the 540 identified genes using gene enrichment (p = 0.01) analysis. Blue bar represent the p-value of the comparison and yellow line represents the change ratio of the pathway. Red bar represent the percentage of genes upregulated inside each pathway in regressing lesions meanwhile green bars represent downregulated ones, yellow line in this graphic represents the p-value of the comparison. All the most significant pathways belong to the immune response and are upregulated in regressing lesions. (b) Antigen presentation plus IRF-1 self organizing network according to IPA analysis based on the 540 identified genes derived from the low stringency analysis. Upregulated genes in regressing metastases are represented in red. Fold-change of the gene is represented by color intensity. The figure shows a consistent activation of the immune genes inside regressing lesions. The analysis did not identify any downregulated gene in the network.

Figure 3

Immunoperoxidase staining of frozen sections of metastases: (a) tumor cells from both progressing and regressing metastases do not express HLA-DR. The staining shows a high degree of HLA-DR positive cellular infiltration in regressing metastasis; in contrast, the progressing lesions have very little infiltration. (b) CD3 staining corresponds to T lymphocyte infiltration (c and d). The number of CD4 positive cells in the infiltrate is higher than the number of CD8 cells.

Figure 4

Normalized mRNA levels in tumor cells isolated by laser microdissection from melanoma patients. The progressing metastases (P) obtained from both patients showed low residual levels of HLA-A, -B and -C loci. In contrast, the regressing tumor cells (R) displayed a high transcription of the tree HLA loci. Regressing metastases obtained after M-VAX treatment in patient M2 showed higher HLA loci expression than those obtained after interferon α 2b.

Figure 5

A common pathway of T cell mediated rejection is showed in different pathological entities.