Prognostic Significance of Gremlin1 (GREM1) Promoter CpG Island Hypermethylation in Clear Cell Renal Cell Carcinoma

Abstract

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P ‫؍‬ 0.02), stage (P ‫؍‬ 0.013), grade (P ‫؍‬ 0.04), tumor (P ‫؍‬ 0.001), and endothelial cell (P ‫؍‬ 0.0001) proliferation and decreased mean vessel density (P ‫؍‬ 0.001) in a hospital-based ccRCC series (n ‫؍‬ 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] ‫؍‬ Clear cell renal cell carcinoma (ccRCC) accounts for Ϸ75% of all cases of renal cell cancer, 1 and is characterized by increased vascularization and an unclear clinical prognosis. Currently, patient performance status, tumor size, nodal and distant metastasis (TNM)-stage, and Fuhrman nuclear grade are the most useful predictors of patient outcome.2 However, interest in additional prognostic molecular markers is growing. Inactivation of the von Hippel-Lindau (VHL) gene has been shown to be a common and early event in the carcinogenesis of ccRCC.3-5 Fifty to 70% of ccRCC tumors harbor a VHL mutation, 5,6 and in 5% to 20% of ccRCCs VHL is silenced by promoter CpG island hypermethylation. 7,8 Although defective VHL functioning is a key event in the development in both sporadic and hereditary ccRCCs, alterations in the structure or regulation of the VHL gene do not Supported by the Kootstra-Talent Fellowship Program, Maastricht

Analysis of Promoter CpG Island Hypermethylation in Cancer: Location, Location, Location!

“…These hypermethylation markers are promising tools to detect cancer cells in tissue and body fluids (6,7) with the use of simple PCR technology (8)(9)(10). Proof of principle for the clinical value of methylation markers has been reported for early detection and classification of cancer (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21), risk assessment and prognosis (19,(22)(23)(24), and prediction of therapy response (25)(26)(27), with some already having shown their importance in (pre)clinical practice. Thus, the promise of methylation changes to become a powerful diagnostic and predictive tool (6) is becoming a reality.…”

Prognostic DNA Methylation Markers for Renal Cell Carcinoma: A Systematic Review

“…Although GREM1 was frequently methylated in RCC, Morris and colleagues [27] could not find an association with patient survival. In contrast, our group and Ricketts' group [29][30][31] Figure 4C). In a subsequent study of Morris' group [32], nine genes were identified to be frequently methylated in primary RCC tumor samples.…”

“…Besides choice of method and cut-off value, also the exact genomic location of the analyzed CpG dinucleotides is important [74]. This was illustrated by van Vlodrop and colleagues [30], who showed that GREM1 methylation varies within the promoter CpG island, with also methylation-region-specific effects on patient outcome.…”

“…Promoter methylation of LAD1 [31,39] and NEFH [31,40] not only predicted patient survival, but also response to treatment with antiangiogenic agents. Conflicting results regarding the prognostic value of promoter methylation of SFRP1 [27][28][29], PCDH8 [29,32,38], GREM1 [27,[29][30] and RASSF1A [26,[35][36][37] were found. Several reasons for these inconsistent results can be adduced.…”

Prognostic and diagnostic relevance of hypermethylated in cancer 1 (HIC1) CpG island methylation in renal cell carcinoma

“…For DAPK-1 as well as for SCUBE3 an association with an increased risk of recurrence has been shown (10,33). Van Vlodrop et al were also able to show significantly worse survival to be associated with methylation of one region within the GREM-1 CGI in univariate analysis and decreased overall survival in correlation with hypermethylation of another GREM-1 CGI subregion in multi-and univariate analysis (34). By creating a combined methylation score for the Wnt-Antagonists sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1 and Dkk-3 Urakami et al instead of were able to show statistical association with a shortened overall survival found SFRP1, BNC1 and COL14A1 to be related with poor prognosis (9,35).…”