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, 11 (1), 161

Molecular and Immune Correlates of TIM-3 (HAVCR2) and Galectin 9 (LGALS9) mRNA Expression and DNA Methylation in Melanoma

Affiliations

Molecular and Immune Correlates of TIM-3 (HAVCR2) and Galectin 9 (LGALS9) mRNA Expression and DNA Methylation in Melanoma

Tobias A W Holderried et al. Clin Epigenetics.

Abstract

Background: The T cell immunoglobulin and mucin-domain containing-3 receptor TIM-3 (also known as hepatitis A virus cellular receptor 2, encoded by HAVCR2) and its ligand galectin 9 (LGALS9) are promising targets for immune checkpoint inhibition immunotherapies. However, little is known about epigenetic regulation of the encoding genes. This study aimed to investigate the association of TIM-3 and LGALS9 DNA methylation with gene expression, patients' survival, as well as molecular and immune correlates in malignant melanoma.

Results: Methylation of all six TIM-3 CpGs correlated significantly with TIM-3 mRNA levels (P ≤ 0.05). A strong inverse correlation (Spearman's ρ = - 0.49) was found in promoter regions, while a strong positive correlation (ρ = 0.63) was present in the gene body of TIM-3. High TIM-3 mRNA expression (hazard ratio (HR) = 0.88, 95% confidence interval (CI) [0.81-0.97], P = 0.007) was significantly associated with better overall survival. Seven of the eight LGALS9 CpG sites correlated significantly with LGALS9 mRNA levels (P ≤ 0.003). Methylation at five CpG sites showed a strong inverse correlation (Spearman's ρ = - 0.67) and at two sites a weak positive correlation (Spearman's ρ = 0.15). High LGALS9 mRNA expression was significantly associated with increased overall survival (HR = 0.83, 95%CI [0.75-0.93], P = 0.001). In addition, we found significant correlations between TIM-3 and LGALS9 methylation and mRNA expression with immune cell infiltrates and significant differences among distinct immune cell subsets.

Conclusions: Our study points toward an epigenetic regulation of TIM-3 and LGALS9 via DNA methylation and might provide an avenue for the development of a predictive biomarker for response to immune checkpoint blockade.

Keywords: Biomarker; DNA methylation; Galectin 9; HAVCR2; Immunotherapy; LGALS9; Melanoma; Prediction; Prognosis; TIM-3.

Conflict of interest statement

Dimo Dietrich owns patents and patent applications on biomarker technologies and methylation of immune checkpoint genes as predictive and prognostic biomarkers (DE 10 2016 005 947.8, DE 10 2015 009 187.5, DE 10 2017 125 780.2, PCT/EP2016/001237). The patents are licensed to Qiagen GmbH (Hilden, Germany). Dimo Dietrich is a consultant of Qiagen. The University Hospital Bonn (PI Dimo Dietrich) receives research funding from Qiagen. The other authors have declared that no conflict of interest exists.

Figures

Fig. 1
Fig. 1
Genomic Organization of the TIM-3 (HAVCR2) (a) and LGALS9 (b) genes. Shown are CG-density and target sites of HumanMethylation450 BeadChip beads. The modified illustration was exported from www.ensemble.org (release 96) and is based on Genome Reference Consortium Human Build 38 patch release 12 (GRCh38.p12). Beads are numbered as follows: cg19110684 (1), cg19646897 (2), cg15371617 (3), cg17484237 (4), cg19063654 (5), cg18374914 (6) (all TIM-3); cg19654781 (7), cg10699049 (8), cg27625456 (9), cg21157094 (10), cg23290146 (11), cg05105919 (12), cg03909504 (13), cg06852032 (14) (all LGALS9)
Fig. 2
Fig. 2
Correlations between TIM-3 and LGALS9 methylation with mRNA expression. Correlations between methylation of CpG site 2 (TIM-3, cg19646897, a) and CpG site 12 (LGALS9, cg05105919, b) with respective mRNA expression in N = 468 melanoma samples
Fig. 3
Fig. 3
TIM-3 methylation in leukocytes, melanocytes, and melanoma cell lines. TIM-3 methylation at six sites in isolated leukocytes (monocytes, granulocytes, B cells, CD8+ T cells, and CD4+ T cells) from healthy donors (N = 28), melanocytes (N = 3), and melanoma cell lines (N = 9), extracted from the Geo Database. Red bars indicate mean methylation values. P-values refer to the Kruskal–Wallis test
Fig. 4
Fig. 4
LGALS9 methylation in leukocytes, melanocytes, and melanoma cell lines. LGALS9 methylation at eight sites in isolated leukocytes (monocytes, granulocytes, B cells, CD8+ T cells, CD4+ T cells) from healthy donors (N = 28), melanocytes (N = 3) and melanoma cell lines (N = 9), extracted from the Geo Database. Red bars indicate mean methylation values. P values refer to the Kruskal–Wallis test
Fig. 5
Fig. 5
Correlations of TIM-3 and LGALS9 methylation and mRNA expression with leukocytes infiltration. Shown are Spearman’s rank correlations (Spearman’s ρ) between methylation/mRNA expression of TIM-3/LGALS9 and leukocyte fraction, as well as tumor infiltrating leukocytes, including lymphocytes (CD8+ T cells, regulatory T cells, T follicular helper cells, T helper cells 1, T helper cells 2, T helper cells 17, γδ T cells, naïve CD4+ T cells, resting and activated memory CD4+ T cells, naïve B cells, memory B cells, plasma cells, resting and activated natural killer cells), monocytes and macrophages (M0/M1/M2 macrophages), resting and activated dendritic cells, resting and activated mast cells, eosinophils, and neutrophils. Immune signatures of tumor infiltrating leukocytes were based on RNAseq analysis and the leukocyte fraction was based on methylation analysis. Only statistically significant (P < 0.05) are shown in color. P values and Spearman’s ρ correlation coefficients can be found in Additional file 1
Fig. 6
Fig. 6
Kaplan–Meier analysis of overall survival in melanoma patients stratified according to TIM-3 (a) and LGALS9 (b) mRNA expression. Patient samples were dichotomized based on optimized cut-offs in N = 468 melanoma patients from The Cancer Genome Atlas. P values refer to the log rank test

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