One of the most significant findings of this large and comprehensive genetic and epigenetic analysis of the VHL
gene in sporadic conventional RCC was the very small numbers of tumours (<5%) with no VHL
involvement. Furthermore these six cases were difficult to classify accurately in most cases due to dedifferentiation. This is consistent with a recent study analysing 205 cases of conventional RCC where 91% of cases had mutation or methylation (34
), and a smaller study where biallelic inactivation of VHL was found in 49/57 (86%) cases (22
) with the corresponding figures for our study being 88% and 86% respectively. Much lower figures have been reported previously in many of the published studies. The higher levels currently detected may be due to the improved sensitivity of mutation detection, greater stringency in tissue selection for nucleic acid extraction and elimination of non-conventional RCC subtypes following pathological review. These findings confirm that that VHL involvement may provide a molecular basis for classification as conventional RCC (49
The rate of LOH (89.2%) observed in our series was comparable with other published data (15
). In 14 samples LOH alone was observed which could potentially be due to mutations outside the region screened in this study such as deep intronic mutations that have an effect on splicing or mutations in the promoter that alter transcription. Alternatively these RCCs may represent a subgroup involving other tumour suppressor genes on chromosome 3p. The mutation rate (74.6%) found in this study was also high, being second only to a recently published paper which employed endonuclease scanning and sequencing for high throughput and sensitive mutation detection and reported mutations in 82.4% of 205 cases (34
). The distribution of our mutations showed a high frequency of mutations around codons 65-76, 86-90 and 158-168 as well as at intron-exon boundaries (114 and 155), consistent with mutations reported in the literature in sporadic conventional RCC. No mutations were found in the first 54 codons of the VHL
gene in this study and in the literature only 54 mutations affecting codons 1-54 have been reported, the majority of which result from three studies (31
). The rate of methylation (31.3%) reported here was notably higher than that found in other studies with most reporting ~5-15% and the previous highest being 20.4% in our initial study (9
). Different methods have been adopted for methylation analysis and different CpG islands analysed and this together with possible differences in sensitivity in the PCR make comparability between studies difficult.
The lack of consistency between studies in terms of associations between VHL changes and clinical variables probably arises from differences in study stringency (methodology, accurate histological subclassification and tissue selection) and the relatively small size of many studies which with multiple testing can lead to false positives which fail to replicate. Few studies have reported on the significance of VHL changes and patient outcome. The presence of VHL
mutations has been associated with improved cancer-free and cancer-specific survival in patients with stage I to III disease treated with radical nephrectomy (p
=0.0024 and p
=0.023); this held for subsets of patients with higher grade (G2 to 4 or G3 and 4) and stage (II and III or III) tumours (46
). The absence of mutation was associated with more aggressive tumours in a study of 100 patients that also examined CAIX expression. High CAIX was associated with VHL
mutation and the data allowed stratification of patients into 3 groups according to the presence of VHL
mutation status and/or strong CAIX expression (36
). Although we did not find these relationships between VHL mutation itself and outcome, our study does suggest that no VHL
involvement confers a worse prognosis than having either methylation or LOH and mutation, although these associations were not statistically significant, possibly given the very small numbers without VHL involvement. Similarly tumours with no LOH or with no VHL involvement were associated with necrosis, higher grade and sarcomatoid features and there was a trend towards the group with mutations potentially encoding the least truncated VHL protein having a worse prognosis, although again this was not significant.
Loss of function mutations have been observed to associate with poor prognosis compared to missense or no mutation (28
). A further study showed no overall association between the presence of mutation and patient characteristics, but did find relationships with the prevalence of particular subtypes of mutation such as between nonsense mutations and grade and nodal status and metastasis (34
). Our study failed to confirm these findings, possibly due to the small number of events in some subcategories, even in a relatively large study. Preliminary studies have also suggested that VHL mutation status correlates with response to VEGF-directed therapies (38
) and corroboration of these observations in independent cohorts is now of great importance.
Examination of the VHL protein itself and downstream proteins/pathways may provide more insight into the functional consequences of particular genetic changes and their clinical relevance. In the case of truncations resulting in early termination, nonsense mediated decay may occur resulting in abrogation of protein expression similar to the potential effect of promoter methylation. Other mutations such as missense mutations or truncations not resulting in nonsense-mediated decay may encode proteins which have different effects on particular VHL functions. A recent study subclassifying conventional RCCs on the basis of the activity of downstream pathways showed that VHL-involved tumours expressing HIF-2α showed increased c-Myc activity whereas RCCs with no apparent VHL
involvement (HIF-α negative) or VHL-involved tumours expressing HIF-1α/HIF-2α showed increased Akt/mTOR and ERK/MAPK signalling (22
). The potential significance of this molecular stratification remains to be determined, but clearly indicates the need to analyse the functional consequences of the VHL changes at the protein level and is an exciting area of research that warrants future investigation.
Statement of translational relevance
The central role played by the VHL tumour suppressor in the development of sporadic conventional RCC has been clearly demonstrated, but the clinical significance of particular VHL defects remains to be determined, with a lack of agreement between initial studies. Larger more stringent studies using robust methods for LOH, mutation and methylation detection applied to good quality tumour samples with more extensive clinical data including clinical outcome are required. This study extends our previous report to 177 patients with conventional RCC. The results indicate VHL gene involvement in almost all conventional RCCs and suggest that subclassification into groups that correlate with prognosis or response to therapy will be made on the basis of the biological effects of specific VHL alterations rather than their presence per se.