Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes

Abstract

BackgroundThe CpG island methylator phenotype (CIMP) of clear cell renal cell carcinomas (ccRCCs) is characterized by accumulation of DNA methylation at CpG islands and poorer patient outcome. The aim of this study was to establish criteria for prognostication of patients with ccRCCs using the ccRCC-specific CIMP marker genes.MethodsDNA methylation levels at 299 CpG sites in the 14 CIMP marker genes were evaluated quantitatively in tissue specimens of 88 CIMP-negative and 14 CIMP-positive ccRCCs in a learning cohort using the MassARRAY system. An additional 100 ccRCCs were also analyzed as a validation cohort.ResultsReceiver operating characteristic curve analysis showed that area under the curve values for the 23 CpG units including the 32 CpG sites in the 7 CIMP-marker genes, i.e. FAM150A, ZNF540, ZNF671, ZNF154, PRAC, TRH and SLC13A5, for discrimination of CIMP-positive from CIMP-negative ccRCCs were larger than 0.95. Criteria combining the 23 CpG units discriminated CIMP-positive from CIMP-negative ccRCCs with 100% sensitivity and specificity in the learning cohort. Cancer-free and overall survival rates of patients with CIMP-positive ccRCCs diagnosed using the criteria combining the 23 CpG units in a validation cohort were significantly lower than those of patients with CIMP-negative ccRCCs (P = 1.41 × 10−5 and 2.43 × 10−13, respectively). Patients with CIMP-positive ccRCCs in the validation cohort had a higher likelihood of disease-related death (hazard ratio, 75.8; 95% confidence interval, 7.81 to 735; P = 1.89 × 10−4) than those with CIMP-negative ccRCCs.ConclusionsThe established criteria are able to reproducibly diagnose CIMP-positive ccRCCs and may be useful for personalized medicine for patients with ccRCCs.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-772) contains supplementary material, which is available to authorized users.

Novel method for DNA methylation analysis using high‐performance liquid chromatography and its clinical application

“…The peaks obtained for the synthetic DNA fragments 5, 6, 7 and 8, which contained 20 gene for discrimination of more aggressive CIMP-positive ccRCC from CIMP-negative ccRCC in our previous study using specimens of tumorous tissue and MassARRAY analysis. 16 After 10 measurements were taken for each fragment, excellent concordance of the retention time for each fragment was revealed ( Figure 2D), indicating the reproducibility of HPLC analysis using the newly developed column. In addition, it was revealed that a difference in methylation level of 5% (ie, 20% vs 25% [P = 4.77 9 10 À11 ] and 25% vs 30% [P = 1.16 9 10 À8 ]) would be statistically distinguishable ( Figure 2D) in a clinically feasible setting.…”

“…In our previous study, 14 ccRCC included in the present study were found to be CIMP-positive using genome-wide DNA methylation analysis by Infinium assay 11 and DNA methylation quantification of the 7 ccRCC-specific CIMP marker genes using the MassARRAY system. 16 Although the present stratification of 16…”

“…To confirm whether this HPLC method is reproducible, we carried out a within‐run reproducibility test using PCR products obtained from Fragments 2, 3 and 4 corresponding to DNA methylation levels of 20%, 25% and 30%, respectively (Table ), which were around the cut‐off value (27.2%) of the DNA methylation level of the FAM150A gene for discrimination of more aggressive CIMP‐positive ccRCC from CIMP‐negative ccRCC in our previous study using specimens of tumorous tissue and MassARRAY analysis . After 10 measurements were taken for each fragment, excellent concordance of the retention time for each fragment was revealed (Figure D), indicating the reproducibility of HPLC analysis using the newly developed column.…”

“…The FAM150A gene is a CIMP marker gene showing a high area under the curve value (0.968) in receiver operating characteristic analysis for discrimination of CIMP-positive ccRRC from CIMPnegative ccRCC. 16 In the present study, 384-bp PCR products encompassing the promoter region of the FAM150A gene from the 98 ccRCC were subjected to HPLC analysis. PCR products from synthetic DNA fragments 1 and 10 with methylation levels of 0% and 100%, respectively (Table 1), were measured simultaneously as control samples.…”

“…Several methods for analysis of DNA methylation (eg, quantitative methylation-specific PCR [MSP], 13 MassARRAY 14 and pyrosequencing) 15 have often been used for quantitative analyses, especially in research. 16,17 Existing methods generally require long analysis times, complex procedures and expensive, large-scale equipment. Therefore, to facilitate dissemination of DNA methylation diagnostics, it is crucial to develop an analytical system capable of delivering data readily, quickly and precisely, especially for diseased cells with DNA methylation abnormalities that need to be discriminated from contaminating cells.…”