Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors

Abstract

Purpose: To provide a comprehensive, thorough analysis of somatic mutation and promoter hypermethylation of the von Hippel-Lindau (VHL) gene in the cancer genome, unique to clear cell renal cancer (ccRCC). Identify relationships between the prevalence of VHL gene alterations and alteration subtypes with patient and tumor characteristics.

Experimental design: As part of a large kidney cancer case-control study conducted in Central Europe, we analyzed VHL mutations and promoter methylation in 205 well-characterized, histologically confirmed patient tumor biopsies using a combination of sensitive, high-throughput methods (endonuclease scanning and Sanger sequencing) and analysis of 11 CpG sites in the VHL promoter.

Results: We identified mutations in 82.4% of cases, the highest VHL gene mutation prevalence reported to date. Analysis of 11 VHL promoter CpG sites revealed that 8.3% of tumors were hypermethylated and all were mutation negative. In total, 91% of ccRCCs exhibited alteration of the gene through genetic or epigenetic mechanisms. Analysis of patient and tumor characteristics revealed that certain mutation subtypes were significantly associated with Fuhrman nuclear grade, metastasis, node positivity, and self-reported family history of RCC.

Conclusion: Detection of VHL gene alterations using these accurate, sensitive, and practical methods provides evidence that the vast majority of histologically confirmed ccRCC tumors possess genetic or epigenetic alteration of the VHL gene and support the hypothesis that VHL alteration is an early event in ccRCC carcinogenesis. These findings also indicate that VHL molecular subtypes can provide a sensitive marker of tumor heterogeneity among histologically similar ccRCC cases for etiologic, prognostic, and translational studies.

Figure 1

Distribution of VHL mutations by codon. A, all mutations; B, mutations with a low RSI value (5−30%).

Figure 2

Analysis of VHL promoter methylation across 11 CpGs among mutation negative (N=16) and mutation positive (N=10) ccRCC DNAs. Methylation status of each CpG is indicated as follows: Yellow=unmethylated, blue=partial methylation, red=fully methylated, and the hatched box indicates that the CpG was uninformative. Positive (methylated) and negative (unmethylated) controls included in each analysis are shown at the bottom and VHL mutation status is shown on the left. Fully and partially methylated CpG sites are summed in the far right column. Cases that had at least 4 methylated CpG sites in the VHL promoter were considered methylation positive.