In the era of cancer immunotherapy, application of immune checkpoint blockade (ICB) targeting cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and/or the programmed cell death 1 (PD-1/PD-L1) axis led to improved clinical outcomes in advanced clear cell renal cell carcinoma (ccRCC).1–3 Both combination of anti-PD-1 and anti-CTLA-44 as well as combined anti-PD-1/PD-L1 plus a tyrosine kinase inhibitor (TKI)5–8 are currently applied as first-line therapy in metastatic ccRCC. As prospective clinical trials comparing these first-line therapies are still pending, both therapy combinations are currently considered equivalent in the intermediate and poor-risk groups defined by the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC). In this context, a robust biomarker for an optimal upfront therapy decision and treatment sequencing in the clinical setting of metastatic ccRCC is missing.1 9

Tumor-intrinsic PD-L1 expression predicts response to anti-PD-1 ICB in various tumor entities, but in ccRCC it is of limited use and the European Association of Urology recommend not to consider this biomarker for patient stratification.10–13 Furthermore, a predictive biomarker that evaluates the effectiveness of an anti-PD-1 blockade is also of limited relevance in ccRCC, as the PD-1/PD-L1 immune axis is targeted in both first-line therapies as the current backbone of first-line ccRCC therapy. Of note, in CheckMate214, the study that ultimately led to the approval of ICB+ICB in ccRCC, only PD-L1 expression was evaluated regarding response rates,4 thereby excluding half of the biological mechanism of this therapy approach, precisely the blockade of the CTLA-4 immune checkpoint.14 A robust predictive biomarker for anti-CTLA-4 monotherapy is also currently lacking despite its high clinical relevance, as several new antibodies and probodies, which promise reduced off-tumor toxicity, are being developed and are already tested in clinical trials (eg, ClinicalTrials.gov Identifier: NCT03369223).15 In this context, we were recently able to provide strong evidence that the methylation status of the CTLA-4 encoding gene CTLA4 predicts response to both anti-PD-1 and anti-CTLA-4 targeted ICB as well as anti-CTLA-4 monotherapy in patients with melanoma.16 17 In the present study, we therefore comprehensively investigated the promoter DNA methylation status of CTLA4 in ccRCC with regard to transcriptional activity, clinicopathological parameters (including survival and response to ICB and TKI), immune cell infiltrates, and an interferon-γ signature. Understanding the epigenetic regulation of CTLA4 in ccRCC is of major interest, as it might be promising as a predictive biomarker to enable a more rational therapeutic decision in favor or against ICB+ICB in patients with ccRCC in the age of individualized therapy.

An epigenetic regulation of the CTLA4 gene via DNA methylation has already been observed in melanoma.16 Interestingly, and of particular clinical interest, the CTLA4 methylation status exhibited a predictive value in patients with melanoma treated with anti-PD-1 plus anti-CTLA-4 immune checkpoint therapy.16 In the current clinical situation for metastatic ccRCC, a robust predictive biomarker for this particular ICB combination therapy, anti-PD-1 plus anti-CTLA-4, is urgently needed as the combination of anti-PD-1/PD-L1 plus TKI is currently considered equivalent and comparative studies are still pending.1 4–6 9 In this study, we therefore comprehensively investigated CTLA4 promoter methylation with regard to transcriptional activity, clinicopathological parameters, and the intratumoral microenvironment in ccRCC tissue. Of note, we observed a strong correlation between the transcriptional activity of CTLA4 and its promoter methylation status in ccRCC. Moreover, CTLA4 promoter methylation and its mRNA expression showed a significant association with the composition of the ccRCC tumor microenvironment: CTLA4 overexpression and concomitant promoter hypomethylation were associated with particularly high lymphocyte infiltration and an increased interferon-γ signature as well as cytolytic activity. CTLA4 hypomethylation thus appears to be a robust surrogate biomarker for an enriched tumor microenvironment. Further, an unfavorable clinical course was evident in primary RCC with hypomethylated CTLA4 promoter and overexpression, respectively. These findings are in line with the literature describing increased immune cell infiltration and immune checkpoint expression in RCC as a negative prognostic marker.26

The transcriptomic and methylation data in the ccRCC TCGA dataset were obtained from whole tumor tissue samples of patients with ccRCC receiving nephrectomy18 19 and are therefore based on the genomic signature of the tumor and its microenvironment, including tumor cells, stroma, infiltrating immune cells, and tumor-associated fibroblasts.27 The complexity of epigenetics is highlighted by widespread tissue-specific and cell type–specific methylation patterns in diverse biological processes;28 however, the characterization of an existing cell line–specific epigenetic regulation of CTLA4 via DNA methylation patterns was not the focus of our study. The aim of our study was to investigate a predictive and whole-tissue based easy-to-implement biomarker for RCC, and excitingly, CTLA4 promoter methylation seems to have cancer-independent predictive potential for ICB response in melanoma and RCC.16 17

As a chemically stable epigenetic modification that is not as dynamic as mRNA or protein expression, DNA methylation patterns represent particularly attractive biomarkers.29 Furthermore, the fact that quantitative and investigator-independent measurement of DNA methylation is even possible in small samples (microdissected cells, liquid biopsies, circulating tumor cells) is a major advantage from the diagnostic point of view.30 31 Basing the data on CTLA4 DNA methylation shown here and the data on CTLA4 methylation in melanoma16 17 strengthen the rationale to test this particular methylation biomarker in clinical trials. In the present study, we have analyzed uncalibrated quantitative methylation levels by means of ß values (Illumina Infinium Technology) and QMS values (qMSP), respectively. These levels, however, do not necessarily reflect true percentage methylation levels. In order to determine percentage methylation, for example, for the transfer of clinically relevant cut-offs to different platforms and assay technologies, absolute methodologies, that is, bisulfite clone sequencing, could be applied.

The CTLA4 promoter hypomethylated ccRCC subgroup was characterized by enhanced immune cell infiltration, in particular, CD8⁺ T cell infiltration indicating these tumors as immunologically “hot tumors". Thus, we asked the question whether the CTLA4 methylation status in treatment-naïve tissue samples prior to initiation of ICB therapy has predictive value to immunotherapy in RCC. Of note, in our multicenter RCC-ICB cohort, CTLA4 promoter hypomethylation predicted ICB treatment success, which also translated into prolonged PFS and OS after ICB treatment initiation, thereby counteracting its negative prognostic value in primary ccRCC at initial diagnosis. CTLA4 methylation status was not associated with TKI response, highlighting that CTLA4 methylation appears to be predictive for immunotherapy only. At initial diagnosis (TCGA and UHB Non-ICB Cohorts), CTLA4 promoter hypomethylation was a negative prognostic biomarker and associated with poor outcome, whereas in metastatic stage prior immunotherapy, it was a favorable biomarker. This is most likely due to its predictive value since the high response to ICB overcompensated the negative prognostic value at initial diagnosis. A similar phenomenon has already been described for melanoma. PD-L1 upregulation is associated with an aggressive subset of melanomas with unfavorable outcome at initial diagnosis but has predictive value for ICB response.32 33 Thus, negative prognostic biomarkers at baseline with strong predictive value for immunotherapy response can overcome their initial negative prognostic value in advanced disease stages. This highlights the potential of CTLA4 methylation as a promising predictive biomarker prior to ICB-treatment initiation in RCC, which has already been suggested for melanoma.16 17

In the current clinical setting of metastatic RCC with multiple first-line therapies, essentially either ICB+TKI or ICB+ICB, there is a tremendous clinical need for robust predictive biomarkers for rational upfront therapy selection, but despite significant efforts, no biomarker that can be easily implemented into clinical practice is available. PD-L1 expression is the only broadly used predictive biomarker, but in ccRCC it is of limited clinical use.10–13 However, patients with ≥1% PD-L1 expression seem to benefit particularly from intensified immunotherapy with nivolumab plus ipilimumab.3 In our multicenter RCC-ICB Cohort, CTLA4 promoter hypomethylation outperformed PD-L1 CPS, which had no significant predictive value in our cohort. Thus, it remains to be prospectively elucidated whether the predictive potential of CTLA4 promoter methylation status will lead to an improved stratification for rational upfront treatment decisions for either ICB+TKI or ICB+ICB.

The main limitations of our study are the retrospective design, the relative small sample size of our RCC-ICB cohort, the heterogeneity of included patients regarding histology (clear-cell and non-clear-cell RCC included), sample origin (primary tumor and distant metastases), and pre-treatment. In order to establish a robust biomarker in this clinical setting, prospective studies are needed to determine the clinical performance of CTLA4 promoter hypomethylation as a predictive biomarker for ICB in patients with ccRCC.

In ccRCC, the important immune checkpoint CTLA-4 is epigenetically regulated by promoter DNA methylation. CTLA4 promoter hypomethylation is a strong biomarker for poor prognosis in patients with ccRCC at initial diagnosis. In contrast, CTLA4 promoter hypomethylation predicted response and favorable outcome to immunotherapy in our multicenter ICB-treated RCC cohort. Thus, it represents a promising candidate for the urgently needed predictive biomarker for optimal upfront treatment decision in metastatic ccRCC.