Identification of a Novel Renal Metastasis Associated CpG-Based DNA Methylation Signature (RMAMS).

Approximately 21% of patients with renal cell cancer (RCC) present with synchronous metastatic disease at the time of diagnosis, and metachronous metastatic disease occurs in 20-50% of cases within 5 years. Recent advances in adjuvant treatment of aggressive RCC following surgery suggest that biomarker-based prediction of risk for distant metastasis could improve patient selection. Biometrical analysis of TCGA-KIRC data identified candidate loci in the NK6 homeobox 2 gene (NKX6-2) that are hypermethylated in primary metastatic RCC. Analyses of NKX6-2 DNA methylation in three gene regions including a total of 16 CpG sites in 154 tumor-adjacent normal tissue, 189 RCC, and 194 metastatic tissue samples from 95 metastasized RCC patients revealed highly significant tumor-specific, primary metastatic-specific, and metastatic tissue-specific hypermethylation of NKX6-2. Combined CpG site methylation data for NKX6-2 and metastasis-associated genes (INA, NHLH2, and THBS4) demonstrated similarity between metastatic tissues and metastatic primary RCC tissues. The random forest method and evaluation of an unknown test cohort of tissues using receiver operator characteristic curve analysis revealed that metastatic tissues can be differentiated by a median area under the curve of 0.86 (p = 1.7 × 10-8-7.5 × 10-3) in 1000 random runs. Analysis of variable importance demonstrated an above median contribution for decision-making of at least one CpG site in each of the genes, suggesting superior informativity for sites annotated to NHLH2 and NKX6-2. Thus, DNA methylation of NKX6-2 is associated with the metastatic state of RCC tissues and contributes to a four-gene-based statistical predictor of tumoral and metastatic renal tissues.

DNA Methylation in INA, NHLH2, and THBS4 Is Associated with Metastatic Disease in Renal Cell Carcinoma.

The detection of DNA methylation in primary tumor tissues could be relevant for early stratification of aggressive renal cell carcinomas (RCCs) as a basis for future personalized adjuvant therapy. Methylated TCGA KIRC based candidate CpG loci in INA, NHLH2, and THBS4 that are possibly associated with RCC metastasis were evaluated by pyrosequencing in 154 paired normal adjacent and primary tumor tissues, as well as in 202 metastatic tissues. Statistical analysis was carried out by bivariate logistic regression for group comparisons, log rank survival analysis, and unsupervised and supervised analysis for the classification of tumors. Increased methylation of INA, NHLH2, and THBS4 loci were significantly associated with distant metastasis in primary tumors (p < 0.05), tissue-specific hypermethylation in metastatic (p = 7.88 × 10-8, 5.57 × 10-10, 2.06 × 10-7) and tumor tissues (p = 3.72 × 10-24, 3.17 × 10-13, 1.58 × 10-19), and shortened progression free survival in patients (p = 0.03). Combined use of CpG site-specific methylation permits the discrimination of tissues with metastatic disease and reveals a significant contribution of CpG sites in all genes to the statistical classification model. Thus, metastasis in RCC is significantly associated with methylation alterations in INA, NHLH2, and THBS4 loci, providing independent information for the potential early detection of aggressive renal cancers as a rationale for stratifying patients to adjuvant therapies.