It has been suggested that the VHL tumor suppressor gene is a major gatekeeper gene for clear cell RCC . About 50%-80% of sporadic RCCs are shown to have mutations of the VHL gene [23, 24]. In this study, which was conducted in Taiwan, the frequency of VHL mutation events for sporadic RCCs was only 20% (3/15), which is much lower than in Western countries. Our results showed promoter hypermethylation in 1 of 15 (6.6%) tumors. This ratio is also lower than that of a previous study in which silencing of the VHL gene by DNA methylation occurred in about 20% of RCCs [10, 23]. Recently, it has been reported in the Cancer Genome Atlas that 7% of clear cell RCCs showed epigenetic silencing at VHL [7, 25]. The discrepancy may be attributed to ethnic effects. However, further studies using larger samples are recommended to verify our results. In the present study, both FHIT and VHL deletions were found in case 13 (age ≥ 50, stage II). A previous study has suggested that FHIT deletion is an early event and VHL deletion as an early and/or late event in RCC .
Dulaimi et al. reported that the frequencies of hypermethylation in 100 kidney tumors were RASSF1A (45%), APC (14%), RARB2 (12%), CDKN2A/p16INK4a (10%), and VHL (8%) . Morris et al. noted that RCCs are most frequently methylated at DAPK (24%), not at RARB2 (0%), CDKN2A/p16INK4a (0%) or CDH13 (3%) . However, these results were not verified by this study as RCCs were found to be most frequently methylated at APC (100%), CDH13 (71.4%, 10/14), CDKN2A (71.4%, 10/14), RARB2 (35.7%, 5/10), and VHL (7.14%, 1/14), not at DAPK1 (0%). Based on the results of this study, frequencies of promoter methylation in RASSF1A, 76.9% (10/13) for RASSF1A_382, and 61.5% (8/13) for RASSF1A_382, in 8/14 cases were much higher than in previous studies in which RASSF1A promoter methylation was detected in 56% and 40% of primary clear cell RCCs, respectively [28, 29]. In this study, neither germline nor somatic DNA methylations in DAPK1 were identified, which is inconsistent with the findings of a previous study . The reasons for this discrepancy are unclear but may be related to the sensitivity of the methods used. With older molecular methods based on radio-labeled primers and polyacrylamide gel electrophoresis, small minor bands may be missed or mistaken. Capillary gel electrophoresis with fluorescence detection allows for the analysis of methylation status with high sensitivity. Dulaimi et al. also noted that RASSF1A methylation is significantly associated with high-grade tumors . Recent studies have highlighted that 16% of RCC cases have loss of CDKN2A through mutation, deletion, or promoter hypermethylation [7, 31].
Although there were differential methylation patterns of the 24 tumor suppressor genes among the 14 RCCs, at least two (mean = 10.7) genes were methylated in each tumor sample. In this study, all RCCs showed methylation of APC specific to RCC, not in normal tissues, which did not change with age. APC gene encodes a 312-kDa protein that acts as an antagonist of the Wnt signaling pathway . Deregulation of Wnt signal pathway through APC deficiency or loss of heterozygosity has recently been implicated in human RCC [33–35]. Aberrant methylation of the APC gene promoter has been reported not only in colon , but also in breast and lung carcinomas . The accumulation of a variety of genetic aberrations is necessary for the initiation and progression of RCCs . These results indicated that methylation of APC is a very early pathognomonic event in tumorigenesis of RCC and can be a candidate diagnostic and therapeutic biomarker as it is found early in the process of carcinogenesis.
In addition to APC methylation, there were a variety of other genetic aberrations. CDKN2B gene methylation was observed in all RCCs, except for case 11. CDKN2B gene on 9p21.3 encodes the p15INK4B protein that binds to and inhibits activation of CDK4 or CDK6 . Germline mutations in CDKN2B have been identified as a novel cause of familial RCC . CASP8 gene encodes Caspase-8 that is an apoptosis-related cysteine peptidase . Methylation at CASP8 has been demonstrated in 16% of RCCs . MLH1 gene encodes proteins that detect and repair DNA mismatches . Expression of mismatch repair proteins MLH1 has been shown to be reduced in 83.7% (118/141) of sporadic RCCs . KLLN gene encodes the protein killin, which is a p53-regulated nuclear inhibitor of DNA synthesis . Bennett el al. found germline methylation in 23/41 (56%) RCC patients and somatic methylation in 19/20 (95%) advanced RCC patients . These results indicated that methylation of APC, CDKN2B, CASP8, MLH1_167, and KLLN is important in the tumorigenesis of RCC, which may inform its diagnostic, clinical, and therapeutic management.