Diversity of the immune microenvironment and response to checkpoint inhibitor immunotherapy in mucosal melanoma
Joris L. Vos, Joleen J.H. Traets, Xiaohang Qiao, Iris M. Seignette, Dennis Peters, Michel W.J.M. Wouters, Erik Hooijberg, Annegien Broeks, Jacqueline E. van der Wal, M. Baris Karakullukcu, W. Martin C. Klop, Arash Navran, Marc van Beurden, Oscar R. Brouwer, Luc G.T. Morris, Mariette I.E. van Poelgeest, Ellen Kapiteijn, John B.A.G. Haanen, Christian U. Blank, Charlotte L. Zuur
Joris L. Vos, Joleen J.H. Traets, Xiaohang Qiao, Iris M. Seignette, Dennis Peters, Michel W.J.M. Wouters, Erik Hooijberg, Annegien Broeks, Jacqueline E. van der Wal, M. Baris Karakullukcu, W. Martin C. Klop, Arash Navran, Marc van Beurden, Oscar R. Brouwer, Luc G.T. Morris, Mariette I.E. van Poelgeest, Ellen Kapiteijn, John B.A.G. Haanen, Christian U. Blank, Charlotte L. Zuur
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Research Article Immunology Oncology

Diversity of the immune microenvironment and response to checkpoint inhibitor immunotherapy in mucosal melanoma

Abstract

Mucosal melanoma (MucM) is a rare cancer with a poor prognosis and low response rate to immune checkpoint inhibition (ICI) compared with cutaneous melanoma (CM). To explore the immune microenvironment and potential drivers of MucM’s relative resistance to ICI drugs, we characterized 101 MucM tumors (43 head and neck [H&N], 31 female urogenital, 13 male urogenital, 11 anorectal, and 3 other gastrointestinal) using bulk RNA-Seq and immunofluorescence. RNA-Seq data show that MucM has a significantly lower IFN-γ signature levels than CM. MucM tumors of the H&N region show a significantly greater abundance of CD8+ T cells, cytotoxic cells, and higher IFN-γ signature levels than MucM from lower body sites. In the subcohort of 35 patients with MucM treated with ICI, hierarchical clustering reveals clusters with a high and low degree of immune infiltration, with a differential ICI response rate. Immune-associated gene sets were enriched in responders. Signatures associated with cancer-associated fibroblasts, macrophages, and TGF-β signaling may be higher in immune-infiltrated, but ICI-unresponsive tumors, suggesting a role for these resistance mechanisms in MucM. Our data show organ region–specific differences in immune infiltration and IFN-γ signature levels in MucM, with H&N MucM displaying the most favorable immune profile. Our study might offer a starting point for developing more personalized treatment strategies for this disease.

Authors

Joris L. Vos, Joleen J.H. Traets, Xiaohang Qiao, Iris M. Seignette, Dennis Peters, Michel W.J.M. Wouters, Erik Hooijberg, Annegien Broeks, Jacqueline E. van der Wal, M. Baris Karakullukcu, W. Martin C. Klop, Arash Navran, Marc van Beurden, Oscar R. Brouwer, Luc G.T. Morris, Mariette I.E. van Poelgeest, Ellen Kapiteijn, John B.A.G. Haanen, Christian U. Blank, Charlotte L. Zuur

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Figure 3

RNA-Seq– and mIF-based characterization of the MucM immune microenvironment across primary sites and compared with CM dataset (21).

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RNA-Seq– and mIF-based characterization of the MucM immune microenvironm...
(A) PCA of primary MucM (N = 101) and CM (N = 64) samples after computational batch correction. (B) Hierarchical clustering heatmap of CM and MucM samples per the Danaher (30) immune cell signatures. (C) Box-and-violin plots visualizing the presence of the 10-gene IFN-γ RNA signature (23) in batch-corrected MucM vs. CM samples. (D) Heatmap leukocyte gene expression signatures in MucM, with hierarchical clustering divides samples into less-infiltrated (violet) and more-infiltrated (magenta) groups. Samples are further annotated with their primary MucM site of origin. (E) Kaplan-Meier overall survival estimate in all patients treated with surgery (N = 86), stratified by immune cluster identified in D. Exact P values were calculated using a 2-sided log-rank test. (F) Stacked bar plot showing the fraction of lower and H&N region MucM clustering as either more or less infiltrated. The P value was calculated using Fisher’s exact test. (G) Box plots showing the Z-scores of CD8+ T cell, cytotoxic cell, and Ayers’ 10-gene IFN-γ signature across MucM regions. (H) Box plots showing the density of CD3+CD8–FoxP3–, CD8+, and CD20+ cells located within the tumor parenchyma, assessed through digital analysis of mIF-stained slides. The CD20+ plot’s y axis was log10-transformed. Dot colors in G and H correspond to the MucM site. Box plots in C, G, and H show medians, IQRs, and whiskers up to 1.5 times the IQR. The violin in C displays the probability density of the data. P values in C and G, and H were calculated using a 2-sided Wilcoxon’s rank-sum test. MucM, mucosal melanoma; CM, cutaneous melanoma; H&N, head and neck; SN, sinonasal; OC, oral cavity, FUT, female urogenital tract; MUT, male urogenital tract; AR, anorectal; GI, gastrointestinal; PCA, principal component analysis.