BTLA promoter hypomethylation correlates with enhanced immune cell infiltration, favorable prognosis, and immunotherapy response in melanoma

Abstract

Background: Immune checkpoint blockade (ICB)-based immunotherapy has significantly improved survival in advanced melanoma. However, many patients exhibit resistance to these therapies. This study examines the impact of BTLA promoter methylation on its expression, immune cell infiltration, and clinical outcomes, evaluating its potential as a prognostic and predictive biomarker for immunotherapy response.

Methods: We analyzed methylation and gene expression data from public datasets (The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO)) and an in-house cohort of melanoma patients treated with ICB therapy at the First Affiliated Hospital of Zhengzhou University. We developed a quantitative methylation-specific PCR (qMSP) assay to measure methylation levels of the cg24157392 and cg03995631 CpG sites, and a targeted bisulfite sequencing assay was used to validate the accuracy of qMSP. We measured BTLA protein expression using multiplex immunofluorescence and immunohistochemical staining methods. Pearson correlation, survival analysis, and immune cell infiltration estimation were conducted to explore the associations between BTLA promoter methylation, mRNA and protein expression, clinical outcomes, and immune characteristics.

Results: Hypomethylation at CpG sites cg24157392 and cg03995631 in the BTLA promoter were significantly associated with higher BTLA mRNA and protein expression. In the TCGA dataset, low methylation at these sites predicted longer overall survival and was validated in an independent cohort of 50 stage III/IV melanoma patients, with an area under the curve of 0.94 for predicting 5-year survival. Furthermore, BTLA promoter hypomethylation correlated with higher infiltration of immune cells, such as CD8+T cells, CD4+T cells, B cells, and macrophages. Additionally, low methylation at cg24157392 and cg03995631, as quantified by the qMSP assay, was significantly associated with better progression-free survival in patients treated with immune checkpoint inhibitors. These findings were further validated using GEO datasets.

Conclusions: BTLA promoter hypomethylation serves as a significant biomarker for favorable prognosis and enhanced response to ICB therapy in melanoma. The developed qMSP assays for cg24157392 and cg03995631 accurately quantified methylation levels and demonstrated their potential for clinical application in patient stratification and personalized immunotherapy.

Keywords: Biomarker; Immune Checkpoint Inhibitor; Skin Cancer; Tumor infiltrating lymphocyte - TIL; Tumor microenvironment - TME.

Figure 1. Overview of CpG sites detected by Infinium HumanMethylation450 BeadChip and genomic structure of BTLA. The illustration (modified) exported from www.ensembl.org (release 113) showing chromosome 3: 112453966-112509472 region, including the BTLA gene and regulatory elements (promoter, promoter flank, enhancer, CTCF binding sites). The two CpG sites (cg24157392 and cg03995631) were located on the promoter region, and the remaining CpG site (cg00477226) was located on exon 5.

Figure 2. Correlations of DNA methylation with BTLA mRNA expression and prognosis in 366 metastatic melanoma patients. (A–C) Pearson correlations of beta value of CpG sites (cg03995631, cg24157392, and cg0047726) with BTLA mRNA expression. (D) Methylation correlation between cg24157392 and cg03995631. (E) The correlation between promoter methylation level (defined as the mean beta value of cg03995631 and cg24157392) and BTLA mRNA expression. (F–G) Kaplan-Meier curves of overall survival in melanoma patients stratified according to median beta values of cg03995631 and cg24157392. (H) Kaplan-Meier curves of overall survival in these patients stratified according to promoter methylation level defined by mean Beta value of two CpG sites. (I) Kaplan-Meier curves of overall survival in these patients stratified according to BTLA mRNA expression.

Figure 3. Correlations of BTLA promoter hypomethylation with high immune cell infiltration and transcriptomic alterations of molecules and pathways in metastatic melanomas. (A) The correlations of BTLA mRNA expression with tumor purity and the infiltration levels of immune cells (B cell, CD8+T cell, CD4+T cell, macrophages, neutrophil and dendritic cells). (B) The comparison of the absolute fraction of TME cells between the high and low promoter methylation groups. (C–E) Box plots show the differences in leukocyte fraction, TIL percentage and cytolytic activity between the two groups. All statistical differences of two groups were compared by Wilcoxon rank-sum test; **p<0.01, ***p<0.001, ****p<0.0001. (F) Volcano showing differentially expressed genes between low and high promoter methylation groups; genes with log2 fold change >1 and p<0.05 are considered to be upregulated in low promoter methylation melanomas. (G) Gene set enrichment analysis plot of hallmark gene sets and pathways enriched (FDR<0.05) in the low promoter methylation group. (H) The correlations between the methylation level of two CpG sites and mRNA expression of immune checkpoint genes. (I–L) Box plots of gene set enrichment scores. Box plots show the median, first and third quartiles, and the whiskers extend to 1.5 times the IQR and p values were calculated using two-sided Wilcoxon rank sum. (I) IFNγ−6. (J) IFNγ expanded immune 18. (K) Effector T cell. (L) IFNγ/Effector T cell. All statistical differences of two groups were compared by Wilcoxon rank-sum test; **p<0.01, ***p<0.001, ****p<0.0001. TIL, tumor-infiltrating lymphocyte; TME, tumor microenvironment.

Figure 4. Validation of correlations of BTLA methylation with prognosis and mRNA expression in independent melanoma cohorts and the FAHZZU cohort treated with immune checkpoint blockade (ICB) therapy. (A–C) The OS analysis based on cg03995631 (A), cg24157392 (B) and promoter (C) methylation level. (D–F) ROC analyses of the cg03995631 (D), cg24157392 (E), and promoter (F) methylation level in predicting 5 years OS. (G–H) Correlations of cg24157392 (G) and cg03995631 (H) methylation with BTLA mRNA expression in the melanoma cohort treated with immunotherapy from Newell’s study. (I–J) Progression-free survival analysis based on cg24157392 (I) and cg03995631 (J) methylation level in the cohort from Newell’s study. (K) Scatter plot showing the negative correlation between BTLA mRNA expression and methylation level of the cg24157392 site as measured by qMSP. Lower methylation levels are associated with higher mRNA expression. (L) Kaplan-Meier survival curve depicting progression-free survival in melanoma patients stratified by high and low methylation levels of cg24157392 as measured by qMSP. Patients with low methylation levels (n=76) had significantly longer progression-free survival compared with those with high methylation levels (n=33) (p=0.012). (M) Scatter plot demonstrating the strong positive correlation between methylation levels of cg24157392 measured by qMSP and targeted methylation sequencing (R=0.92, p=7.9e–13), validating the accuracy of qMSP for assessing methylation at this site. (N) Scatter plot showing the negative correlation between BTLA mRNA expression and methylation level of the cg03995631 site as measured by qMSP. Lower methylation levels are associated with higher mRNA expression. (O) Kaplan-Meier survival curve depicting progression-free survival in melanoma patients stratified by high and low methylation levels of cg03995631 as measured by qMSP. Patients with low methylation levels (n=85) had significantly longer progression-free survival compared with those with high methylation levels (n=24) (p=0.035). (P) Scatter plot demonstrating the strong positive correlation between methylation levels of cg03995631 measured by qMSP and targeted methylation sequencing (R=0.91, p=6.1e−12), validating the accuracy of qMSP for assessing methylation at this site. AUC, area under the curve; FAHZZU, First Affiliated Hospital of Zhengzhou University; OS, overall survival; qMSP, quantitative methylation-specific PCR; ROC, receiver operating characteristic.

Figure 5. Validation of the negative association of BTLA promoter methylation with protein expression and immune cell infiltration. (A) BTLA/CD45RO dual fluorescence immunostaining confirms the colocalization of BTLA (yellow signal) and CD45RO (red signal) within T cells. (B) Representative images show H&E staining of tissue and cell sections and BTLA immunohistochemical (IHC) staining for high and low methylation groups. 20×, 20-fold original magnification; 80×, 80-fold original magnification. (C) Box plot shows the difference of BTLA positive cell fractions between low and high methylation groups. Wilcoxon rank-sum test; *p<0.05. (D) Pearson correlation between promoter methylation and BTLA positive cell fractions by IHC. (E) Box plot shows the difference of systemic immune-inflammation index (SII) between low and high methylation groups. Wilcoxon rank sum one-sided test. (F) The Venn diagram shows the distribution of patients in the high methylation and low methylation groups between the high SII and low SII groups (left panel: cg24157392; right panel: cg03995631). (G) The Venn diagram also illustrates the distribution of patients in the high methylation and low methylation groups across different tumor stages (left panel: cg24157392; right panel: cg03995631).

Figure 6. The positive correlations of BTLA promoter methylation with DNMT3A/B gene expression. (A) Box plots show the gene expression differences of DNMT3A/B between high and low promoter methylation groups in 366 TCGA metastatic melanoma. (B) Pearson correlations between BTLA promoter methylation level and DNMT3A/B gene expression levels in 366 TCGA metastatic melanomas. (C) Pearson correlations between BTLA promoter methylation level (qMSP) and DNMT3A/B gene expression levels (qPCR) in 50 metastatic melanomas from our FAHZZU cohort. FAHZZU, First Affiliated Hospital of Zhengzhou University; qMSP, quantitative methylation-specific PCR; qPCR, quantitative PCR; TCGA, The Cancer Genome Atlas.All statistical differences of two groups were compared by Wilcoxon rank-sum test; ****p<0.0001.