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

SIMPLE SUMMARY: Early prediction of aggressive cancer using biomarkers is thought to be important for future improvements in the personalized treatment of renal cell carcinoma (RCC). Epigenetic alterations, such as DNA methylation, are promising candidates because they are frequently associated with distant metastasis of tumors and combinations of them offer greater informativity. Here, we describe the indication of the metastatic disease state of renal cell tumors by the methylation of three new candidate genes, INA, NHLH2, and THBS4. The inclusion of the methylation status of these genes could improve the early detection of metastatic tendency in renal tumors and help identify patients who will benefit from adjuvant treatments. ABSTRACT: 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.