www.nature.com/bjc ARTICLE Molecular Diagnostics Apelin and apelin receptor expression in renal cell carcinoma Yuri Tolkach1, Jörg Ellinger2, Anika Kremer1, Laura Esser1, Stefan C. Müller1, Carsten Stephan3, Klaus Jung4, Marieta Toma1, Glen Kristiansen1 and Stefan Hauser2 BACKGROUND: The APLNR (apelin receptor) has been shown to be an essential gene for cancer immunotherapy, with deficiency in APLNR leading to immunotherapy failure. The aim of this study is to investigate the expression of APLN (apelin) and APLNR in patients with renal cell carcinoma (RCC), and its association with clinicopathological parameters and survival. METHODS: Three well-characterised patient cohorts with RCC were used: Study cohort 1 (clear-cell RCC; APLN/APLNR mRNA expression; n = 166); TCGA validation cohort (clear-cell RCC; APLN/APLNR mRNA expression; n = 481); Study cohort 2 (all RCC subtypes; APLNR protein expression/immunohistochemistry; n = 300). Associations between mRNA/protein expression and clinicopathological variables/patients’ survival were tested statistically. RESULTS: While APLN showed only very weak association with tumour histological grade (TCGA cohort), APLNR/mRNA protein expression correlate significantly with ccRCC aggressiveness. APLNR is expressed in tumour vasculature and tumour cells at different levels, and these expression levels associate with tumour aggressiveness in opposing directions. APLNR expression was negatively correlated with PD-L1 expression by tumour cells in a subset of patients with ccRCC. APLNR expression in either compartment is an independent prognostic factor for survival of patients with ccRCC. CONCLUSION: The APLNR/APLN-system appears to play an important role in ccRCC, warranting further clinical investigation. British Journal of Cancer (2019) 120:633–639; https://doi.org/10.1038/s41416-019-0396-7 BACKGROUND tissue bank at the CIO Cologne-Bonn (clinical characteristics in Metastatic renal cell carcinoma (RCC) is a common oncological Suppl. Table 1). Patients underwent radical or partial nephrect- disease worldwide, that is resistant to radiation therapy omy at the Department of Urology at the University Hospital and conventional cytotoxic chemotherapy.1 Immunotherapy has Bonn. been a viable treatment since the 1990s, but the advent of immune Fresh-frozen tissues were stored at −80 °C prior to use. Total checkpoint inhibition has improved therapy responses consider- RNA was isolated from 166 ccRCC and 102 normal renal tissue ably.2,3 Predictive biomarkers for rational patient selection are samples as described before.13 In brief, total RNA was isolated with currently under extensive investigation.4 the mirVana miRNA Isolation Kit (Ambion, Foster City, CA, USA) Recently, the apelin receptor (coded by the APLNR gene) was and subsequently treated with DNase (DNA-free Kit, Ambion). The identified as prerequisite for successful cancer immunotherapy, RNA quantity was determined using a NanoDrop 2000 spectro- with APLNR deficiency associated with immunotherapy failure.5 photometer (Thermo Scientific, Wilmington, DE, USA). RNA Since apelin (APLN) and the apelin receptor have long been known integrity was confirmed by evaluation of the 28S and 18S rRNA to play important roles in vascular physiology,6,7 emerging bands in a gel electrophoresis. evidence of their importance in oncological diseases is not The APLN and APLNR mRNA expression levels were determined surprising.8–11 Given the paucity of data concerning the role using quantitative real-time PCR; ACTB, GAPDH and PPIA were used of APLN and APLNR in renal cell carcinoma12 and the importance as reference genes (see Supplementary Table 2 for primer of immune evasion mechanisms of this tumour, our study aimed to sequences). Relative expression levels were calculated using the investigate the expression of APLN and APLNR in RCC and its 2-ΔΔCT algorithm using the Applied Biosystems QuantStudio 3D association with clinicopathological parameters and survival. Analysis Suite Cloud software. cDNA was synthesised from 1 µg total RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Saint-Germain-en Laye, France). For each qPCR, MATERIALS AND METHODS 5 ng/µl cDNA was amplified with 1× SYBR Premix Ex Taq II and mRNA analysis (Study cohort 1) ROX Plus with 10 pmol/µl forward/reverse primer. PCR With pre-operative written informed consent, renal tissues experiments were performed on the QuantStudio 5 Real-Time were collected from 166 patients within the framework of the PCR System (Thermo Fisher Scientific). 1Institute of Pathology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany; 2Department of Urology, University Hospital Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany; 3Department of Urology, Charite - Universitätsmedizin Berlin, Schumannstrasse 20, 10117 Berlin, Germany and 4Berlin Institute for Urologic Research, Schumannstrasse 20, 10117 Berlin, Germany Correspondence: Glen Kristiansen ([email protected]) or Stefan Hauser ([email protected]) These authors contributed equally: Yuri Tolkach, Jörg Ellinger. Glen Kristiansen and Stefan Hauser shared senior authors. Received: 2 May 2018 Revised: 14 January 2019 Accepted: 21 January 2019 Published online: 20 February 2019 © Cancer Research UK 2019 Apelin and apelin receptor expression in renal cell carcinoma Y Tolkach et al. 634 The Cancer Genome Atlas analysis (TCGA cohort) SC-47778, 1:200) was used as a loading control, probed with Clinical data and normalised mRNA expression data generated HRP-conjugated rabbit anti-mouse secondary antibody (Dako, with Illumina HiSeq 2000 RNA sequencing platform, version 2 P-0260, 1:5,000); for results see Fig. 3 and Suppl. Data 1. (data version 28.01.2016) were extracted from TCGA for patients with clear-cell RCC. After database construction with thorough Microvessel density assessment control of data quality, 481 patient cases with complete mRNA All TMA slices were digitalised at an objective magnification of expression and clinical information were available for analysis. 20× and saved in MIRAX-format. Automated microvessel density assessment was carried out using “Vessel detection” module in Immunohistochemistry analysis (Study cohort 2) Tissue Studio of Definiens Developer XD software (v.2; Munich, Three-hundred patients diagnosed with renal cell carcinoma after Germany) on individual tumour spots (Suppl. Figure 2). Median radical or partial nephrectomy at a single institution (Department microvessel densities (number of vessels/mm2) were calculated of Urology, Charité—Universitätsmedizin Berlin, Germany; when more than one tumour spot was available for analysis. 1992–2004) were included in this study (Suppl. Table 1). The mean follow-up time was 117 months (total range: 1–267 months), Ethical issues allowing the calculation of overall survival as an endpoint. The study was approved by the ethic committee at the University Formalin-fixed, paraffin-embedded archive tissue was used to Bonn (vote: 317/17). construct a tissue microarray (TMA) with two tumours and two normal tissue spots (diameter 1 mm) from every patient. The Statistical analyses tissue microarray was cut (3 µm thick) and mounted on superfrost All statistical analyses were made in R (version 3.2.3; The R slides (Menzel Gläser, Brunswick, Germany). After deparaffinisation Foundation for Statistical Computing). The follow-up period for with xylene and gradual rehydration, antigen retrieval was overall survival analyses in the immunohistochemistry cohort achieved by pressure-cooking in 0.01 mol/L citrate buffer for 5 (study cohort 2) was limited to 180 months. Optimal cut-off values min. Slides were incubated with primary antibody (APLNR rabbit for mRNA expression data were determined using the survMisc polyclonal antibody, ThermoFisher Scientific, Catalogue number package for R (based on consecutive evaluation of all available PA5-21285; Dilution 1:50). The slides were counterstained with cut-offs using univariate Cox regression). haematoxylin and aqueously mounted. The immunohistochemical staining was evaluated blind to clinical outcome, clinical and 1234567890();,: pathological stage. Staining intensities were graded separately for RESULTS cytoplasm and membrane of tumour cells and endothelial cells of mRNA expression analyses (mRNA cohort) tumour vessels. A 4-tier grading system (0: negative; 1: weakly APLN (p = 0.110) and APLNR (p = 0.105) mRNA expression levels positive; 2: moderately positive; 3: strongly positive) was used. were similar in normal (n = 102) and malignant tissues (ccRCC, TMA Slides were also stained with CD34 antibody (monoclonal n = 166) (Fig. 1). APLNR expression was inversely correlated with antibody, Dako/Agilent; Dilution 1:100, m7165) for microvessel histological grade of the tumour (Pearson’s r = −0.17, p = 0.027), density assessment and with PD-L1 antibody (monoclonal mouse pT-stage (Pearson’s r = −0.20, p = 0.009) and presence of meta- anti-human antibody, Clone 22C3, Dako/Agilent; pharmDx kit). PD- static disease (Pearson’s r = -0.20, p = 0.009), while APLN mRNA L1 staining was evaluated by means of the percent of positive expression showed no significant correlation. tumour cells in a TMA spot (membrane staining). In the survival analysis (n = 154, number of events: OS = 31, CSS = 21) APLN expression showed no prognostic association with Cell lines either overall survival or cancer-specific survival. In contrast, APLNR The following cell lines were used for western blot: Caki-1 (human was predictive for overall (OS) and cancer-specific