An immune-active tumor microenvironment favors clinical response to ipilimumab

Abstract

Purpose: Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized.

Experimental design: Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3 weeks after the start of treatment in a phase II clinical trial.

Results: Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab.

Conclusions: These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.

Fig. 1

Characterization of genes differentially expressed in pre-treatment tumors between the CA and No-CA groups. The Ingenuity IPA tool was used to characterize the 194 differentially expressed probe sets. a Top biological functions clustering the differentially expressed genes. The P value indicates the significance of the enrichment of input genes in each functional category. b Top canonical pathways clustering the differentially expressed genes. The P value (blue bar) indicates the significance of the enrichment of input genes in each pathway. The ratio (orange square) is the number of genes from the input list that were annotated to the pathway to the total number of genes annotated to the pathway. c, d Heatmap of the expression of the top 26 potential predictive tumor biomarkers. The tumors are grouped by time, before (c) and after (d) treatment with ipilimumab, and by CA status. Color indicates expression level, from low (blue) to high (red)

Fig. 2

Characterization of genes with increased expression after ipilimumab treatment. The Ingenuity IPA tool was used to characterize the 470 probe sets with increased expression after treatment with ipilimumab. a Top biological functions clustering the genes. The P value indicates the significance of the enrichment of input genes in each functional category. b Top canonical pathways clustering the genes. The P value (blue bar) indicates the significance of the enrichment of input genes in each pathway. The ratio (orange square) is the number of genes from the input list that were annotated to the pathway to the total number of genes annotated to the pathway. c, d Heatmap of the expression of the top 29 potential early predictive tumor biomarkers. The tumors are grouped by time, before (c) and after (d) treatment with ipilimumab, and by CA status. Color indicates expression level, from low (blue) to high (red)

Fig. 3

Characterization of genes with decreased expression after ipilimumab treatment. The Ingenuity IPA tool was used to characterize the 269 probe sets with decreased expression after treatment with ipilimumab. a Top biological functions clustering the genes. The P value indicates the significance of the enrichment of input genes in each functional category. b Top canonical pathways clustering the genes. The P value (blue bar) indicates the significance of the enrichment of input genes in each pathway. The ratio (orange square) is the number of genes from the input list that were annotated to the pathway to the total number of genes annotated to the pathway. c–f Heatmap of selected genes with decreased expression in post-treatment tumors from patients of the CA group. Figure shows expression of melanoma-associated markers before (c) and after (d) the treatment, and genes associated with cell proliferation before (e) and after (f) the treatment. Color indicates expression level, from low (blue) to high (red)

Fig. 4

Association of gene expression with TLI and survival. TLI of post-treatment samples was scored after H&E staining of FFPE tumor biopsy slides on a scale of 0–4 in 0.5-unit increments. a Association between gene expression and TLI. The anti-log RMA expression values for a representative probe set were plotted against the TLI scores from the same tumors, in patients showing either CA (filled circle) or No-CA (open circle). b H&E staining of pre- and post-treatment tumor biopsies. i and ii are pre- and post-treatment lymph node samples, whereas iii and iv are pre- and post-treatment skin samples. Melanoma cells are characterized by abundant cytoplasm, large and central nuclei, apparent nucleolus (large arrows), and melanin pigment (star) associated with mononuclear leukocytes (small arrows). An increase in infiltrating mononuclear leukocytes is evident in post-treatment samples (ii and iv). c Association between overall survival and post-treatment gene expression. For each of the representative genes, patients were grouped as having either equal to or greater than (broken lines), or less than (continuous lines) the mean expression of the indicated gene