PMC

TNFRSF9 methylation in distinct cell types.TNFRSF9 methylation at twelve CpG sites within TNFRSF9 in primary melanoma and melanocyte cell lines as well as in isolated PBMCs, monocytes, B cells, CD8⁺ T cells, and CD4⁺ T cells from healthy donors. All P < 0.001 (Kruskal–Wallis test).

Kaplan–Meier analysis of overall survival in melanoma patients stratified according to TNFRSF9 methylation and mRNA expression. Patient samples were dichotomized based on optimized cutoffs. Analysis of CpG sites targeted by beads 3 and 4 was omitted, as generation of an optimized cutoff did not result in significant survival differences. Follow-up data was available from N = 448 (mRNA expression) and N = 450 (methylation) patients, respectively. P-values refer to log-rank tests.

Genomic organization of the TNFRSF9 gene. Shown are regulatory elements, CG-density and target sites of HumanMethylation450 BeadChip beads and the quantitative methylation-specific PCR (qMSP assay). The modified illustration was exported from www.ensemble.org (release 98) and is based on Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13). Beads are numbered as follows: cg16839093 (1), cg27305704 (2), cg18859763 (3), cg07836592 (4), cg23959705 (5), cg06956444 (6), cg14614416 (7), cg18025409 (8), cg14153654 (9), cg08840010 (10), cg17123655 (11), cg16117781 (12).

TNFRSF9 methylation and mRNA expression with regard to response to anti-PD-1 immunotherapy. TNFRSF9 mRNA expression levels in samples from a recently published anti-PD-1-treated cohort comprised of N = 49 responding patients (partial, mixed, and complete), N = 56 non-responding patients, and N = 16 patients with stable disease . TNFRSF9 methylation at CpG targeted by bead 12 in melanoma samples from N = 19 responding (complete and partial responses) and N = 29 non-responding patients. P-values refer to Wilcoxon Mann–Whitney U and Kruskal–Wallis test, respectively. Mean levels are indicated by bars.

Kaplan–Meier analysis of progression-free survival in two cohorts of anti-PD-1 treated melanoma patients stratified according to TNFRSF9 methylation and mRNA expression. Methylation of CpG site targeted by bead 12 was investigated in a case-control study comprised of N = 19 patients with progressive disease and N = 29 patients with response (partial and complete), respectively, to PD-1 directed immunotherapy. TNFRSF9 mRNA expression was evaluated in N = 121 samples from a recently published cohort of patients who received anti-PD-1 directed immunotherapy . Patient samples were dichotomized based on optimized cutoffs. P-values refer to log-rank tests.

Correlation of TNFRSF9 methylation and mRNA expression with immune cell infiltrates. Shown are Spearman's ρ correlation coefficients of significance (P<0.05) between TNFRSF9 methylation and mRNA expression with leukocyte fraction (mRNA: N = 468; methylation: N = 470) and distinct immune cell infiltrate signatures (mRNA: N = 468; methylation: N = 469). Immune cell infiltrates include RNA signatures of lymphocytes (including naive B cells, memory B cells, naive CD4⁺ T cells, activated and resting CD4⁺ memory T cells, T follicular helper cells, regulatory T cells, CD8⁺ T cells, γδ T cells, activated and resting NK cells, and plasma cells), macrophages (including monocytes and M0/M1/M2 macrophages), dendritic cells (including resting and activated dendritic cells), mast cells (including activated and resting mast cells), CD4⁺ T cells (including naive, activated memory, and resting memory CD4⁺ T cells), eosinophils, and neutrophils. P-values and Spearman's ρ correlation coefficients can be found in Supplemental Table 1; n.s.: not significant.