CTLA4 promoter hypomethylation is a negative prognostic biomarker at initial diagnosis but predicts response and favorable outcome to anti-PD-1 based immunotherapy in clear cell renal cell carcinoma

Abstract

Background: In metastatic clear cell renal cell carcinoma (ccRCC), different combination therapies, each including anti-PD-1 immune checkpoint blockade (ICB), are applied as first-line treatment. Robust predictive biomarkers for rational upfront therapy decisions are lacking, although they are urgently needed. Recently, we showed that CTLA4 promoter methylation predicts response to ICB in melanoma. Here, we aimed to investigate CTLA4 methylation in ccRCC and its utility to serve as a predictive biomarker for anti-PD-1 based ICB in metastatic ccRCC.

Methods: CTLA4 methylation was analyzed with regard to transcriptional gene activity (mRNA expression), intratumoral immune cell composition, and clinical course in two ccRCC cohorts obtained from The Cancer Genome Atlas (TCGA cohort, n=533) and the University Hospital Bonn (UHB Non-ICB Cohort, n=116). In addition, CTLA4 methylation as well as CD8⁺ T cell infiltrates and PD-L1 expression were evaluated in pre-treatment samples from a multicenter cohort (RCC-ICB Cohort, n=71). Patients included in the RCC-ICB Cohort were treated with either first line anti-PD-1 based combination therapy (n=25) or monotherapy post-tyrosine kinase inhibition in second line or later. Analyses were performed with regard to treatment response according to RECIST, progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) following treatment initiation.

Results: CTLA4 promoter hypomethylation was significantly correlated with CTLA4 mRNA expression, lymphocyte infiltration, and poor OS in both primary ccRCC cohorts (TCGA: HR 0.30 (95% CI 0.18 to 0.49), p<0.001; UHB Non-ICB: HR 0.35 (95% CI 0.16 to 0.75), p=0.007). In contrast, CTLA4 promoter hypomethylation predicted response and, accordingly, favorable outcomes (PFS and OS) in patients with ICB-treated ccRCC, overcompensating the negative prognostic value of CTLA4 hypomethylation at initial diagnosis. Moreover, in multivariable Cox regression, CTLA4 promoter hypomethylation remained an independent predictor of improved outcome in ICB-treated ccRCC after co-adjustment of the International Metastatic Renal Cell Carcinoma Database Consortium score (HR 3.00 (95% CI 1.47 to 6.28), p=0.003).

Conclusions: Our study suggests CTLA4 methylation as a powerful predictive biomarker for immunotherapy response in metastatic RCC.

Keywords: biomarkers; immunotherapy; kidney neoplasms; tumor; urologic neoplasms.

Figure 1

(A) Genomic organization of the CTLA4 gene and target sites of the Human Methylation450 BeadChip (CpG1: cg08460026 and CpG2: cg05074138) and of the quantitative methylation-specific PCR (qMSP). The illustration (modified) was taken from Ensembl release 104 and is based on Genome Reference Consortium Human Build 38 patch release 13 (GRCh38.p13). (B–D) CTLA4 mRNA expression and promoter methylation status of CpG1 and CpG2 in normal adjacent tissue (NAT) vs clear cell renal cell carcinoma (ccRCC). (E, F) Scatter plots representing CTLA4 promoter methylation in relation to CTLA4 mRNA expression.

Figure 2

Correlation heatmaps visualize Spearman’s ρ correlation coefficients of both the CTLA4 mRNA expression and the promoter methylation status (CpG1+2) with respect to the intratumoral immune cell composition (A), the IFN-γ response, and cytolytic activity (B), respectively. APC, antigen-presenting cell.

Figure 3

Association of the CTLA4 mRNA expression and promoter methylation of CpG1 with respect to the metastatic status (M stage, A, D) and the clinical endpoints event-free survival (EFS, B, E) and overall survival (OS, C, F) in the ccRCC TCGA cohort are depicted. ccRCC, clear cell renal cell carcinoma.

Figure 4

(A) CTLA4 promoter hypomethylation is associated with high lymphocyte infiltration, especially CD8⁺ T cells. (B+C) CTLA4 promoter hypomethylation was associated with unfavorable event-free (EFS) and overall survival (OS). (D) In pre-treatment RCC samples, CTLA4 promoter hypomethylation predicts immune checkpoint blockade (ICB)–treatment response and is associated with prolonged progression-free survival (PFS) after ICB-treatment initiation (E) and favorable OS (F).