Trichloroethylene exposure and somatic mutations of the VHL gene in patients with Renal Cell Carcinoma

Renal cell carcinoma (RCC), the most frequent malignancy in the adult kidney, is usually sporadic [1]. The phenotype is extremely heterogeneous and several classifications of renal epithelial tumours have been proposed [2, 3]. Main histological subtypes of renal epithelial tumours include clear-cell RCC (75%), papillary RCC (10–15%), chromophobe RCC (5%) and oncocytomas (5%). Inactivation of the VHL tumour suppressor gene is thought to result both in development of tumors in the von Hippel-Lindau (VHL) disease (MIM #19330) and in sporadic clear-cell RCC [4]. Germline mutations of VHL gene are responsible for VHL disease, a rare dominantly inherited cancer syndrome predisposing to a number of highly vascularized tumors including multiple clear-cell RCC whereas somatic mutation or methylation of VHL gene is a frequent event in sporadic clear-cell RCC [4, 5]. Trichloroethylene (TCE) is a solvent used in numerous industries, as degreaser in the metal manufacturing industry, solvent for oils and resins, or the production of rubber and as a chemical intermediate in the production of refrigerants. A number of epidemiological studies have investigated the association between exposure to trichloroethylene and renal cell cancer but the results have been inconsistent [6]. A cohort study conducted in a cardboard factory and a case-control study conducted in the same area in Germany found significantly elevated risk for renal cell cancer and trichloroethylene occupational exposure [7, 8]. For subjects in that case control study, a specific pattern of mutations in the VHL gene has been reported in RCC cases with former prolonged and high-level exposures to trichloroethylene [9]. However, another study carried out in Germany to evaluate the phenotype and the genotype of renal tumours in occupationally TCE-exposed patients revealed no unique phenotype, genotype or mutation pattern in the VHL gene of renal tumours after TCE exposure [10].

In France, the results of a case-control study performed in a geographic area with a high frequency and intensity of exposure suggested an association between exposures to high levels of TCE and increased risk of RCC [6, 11, 12]. Thus it seemed to be of interest to further expand this case-control study with a molecular analysis to confirm or not the specific pattern of VHL mutations associated with trichloroethylene exposure reported in the German study. The objective of the study was to test the hypothesis of an association between exposure to trichloroethylene and VHL mutations and the subsequent risk for RCC.

It has been postulated that sporadic and familial renal cell carcinomas have a common carcinogenic pathway and that at least one gene should be altered in both forms. This has been confirmed with the cloning of the VHL gene and the identification of germline and somatic mutations of this gene in VHL patients and sporadic RCC. Today, more than 400 germline mutations have been reported in VHL patients and about 300 somatic mutations in sporadic RCC [13]. The collection of a large number of mutations is necessary to identify key residues in the biological function of the protein, for molecular epidemiology and to establish correlations between the localization of the mutation and specific phenotypes. In a previous study, Beroud et al. [14‐16] have shown that the somatic and germline mutational events are different with only 22% of missense mutations in sporadic RCC vs 63% in familial cases (p < 0.001). They also showed that the distribution of missense mutations is different in the two groups with 68% of missense mutations corresponding to transversion in somatic mutations vs 35% in germline mutations (p < 0.001). Since it is thought that transversions implicate an extrinsic factor, these data support the hypothesis of the involvement of environmental factors in the aetiology of sporadic RCC [17‐19]. Brüning et al. studied the RCC incidence in individuals with former prolonged and high-level exposures to TCE [20]. They found a specific pattern of VHL somatic mutations with a high frequency in exon 2. In 1999, Brauch et al. completed these data and showed multiple intragenic mutations within the same tumour (42% of cases), a very high proportion of C>T transitions (70%) and a hot spot of mutations at codon 81 (p.Pro81Ser) [9]. Altogether these data suggest a role of TCE or its metabolites in these mutational events. To explore this further, we have compared the distribution of somatic VHL mutations in RCC from patients exposed to TCE (from Brüning [20] and Brauch [9]) with the RCC somatic mutations in the UMD-VHL database [13] that collects all published mutations of the VHL gene. The purpose of this analysis was to compare specific pattern of VHL mutations thought to be associated with TCE exposure with a wider collection of VHL gene information.

This comparison confirms that the specific pattern of mutations identified by Brüning [20] and Brauch [9] is different from the pattern in the large UMD-VHL database of somatic VHL mutations in RCC tumours. In fact, this somatic mutation pattern suspected to be associated with TCE exposures is much closer to the germline mutation profile with an excess of missense mutations. In addition, 50% (16 out of 32) of these missense mutations are C>T transitions with 13 involving codon 81. As these transitions do not involve a CpG dinucleotide, we can suspect that they result from the exposure to a toxic substance or any other carcinogen.

In addition the distribution of somatic mutations reveals that the codon 81 has been involved in only one tumour from studies other than those of Brüning et al [20] and Brauch et al. [9]. Overall mutations at this position could be specifically associated to exposure to trichloroethylene. It is however surprising, considering the intensive use of TCE all over the world, that this specific mutation has not been reported in other case series. In our series, no mutation was detected at this particular codon, indicating that, as observed by Schraml et al [10], we have not confirmed the results of Brüning et al. [20] and Brauch et al. [9].

The present study was performed in blinded test fashion regarding TCE exposure data. The series comprised 48 cases of confirmed clear cell RCC. All of these cases had been included in a case control study previously published. 26 of the tumours were Bouin's solution-fixed, 17 were formalin-fixed and 5 were frozen tumours. As reported in the literature, molecular analysis based on archival tissues is possible however often difficult [21]. Actually, formic acid contained in formalin solution, fixation time and period of storage of the tissue blocks often affect the quality of DNA. Furthermore, picric acid contained in Bouin's solution is known to degrade nucleic acids, thus a low yield of PCR amplification of DNA could be obtained. Here 26 tumours (21 fixed and 5 frozen tumours, ie 54%) were successfully analyzed regarding the VHL gene. However, codon 81 was seen for 80% of the cases of clear cell RCC who had been exposed to TCE. Three mutations were detected in the fixed tumours, within exon 1 (c.332G>A, p.Ser111Asn), exon 2 splice site (c.463+2T>C) and exon 3 (c.506T>C, p.Leu169Pro). These three cases were fixed in formalin solution. No mutation was found in the samples fixed in Bouin's solution. Regarding the 5 frozen tumours, one mutation was detected at the exon 2 splice site (c.463+1G>C). The 4 mutations detected in the present study have been described in previous VHL studies: they are reported between 1 and 4 times either at the germline or somatic level in the VHL Mutation Database [25].

Interestingly, it is reported in the literature that a VHL somatic mutation is detected in approximately half of sporadic clear cell RCC [14, 22, 23]. Thus, among the 48 clear cell RCC tumours analyzed in the present study, at least 20 samples would be expected to carry a VHL mutation compared to 4 mutations detected here. However, these 4 mutations were detected in a series of 26 clear cell RCC for which the VHL gene was entirely sequenced, corresponding to 15%. As a number of fixed samples could not be successfully PCR amplified because of DNA degradation, we cannot exclude that some of these samples carry a VHL mutation. However, it was recently suggested a new cause of occupational cancer where there was a molecular analysis of VHL without mutation detected in this gene, suggesting that other genes may be implicated in RCC and in particular linked to chemical exposure [24].

The VHL gene can be inactivated somatically, besides loss of heterozygosity, either by mutation or promoter hypermethylation. The quality of the DNA extracted from fixed tumours did not allow us to analyze the methylation status of the VHL promoter. Thus, we cannot exclude that some tumours may involve hypermethylation.

In the present study, 25 (52%) of the clear cell RCC cases concerned patients who had been exposed to TCE, 12 (48%) of them had been exposed to high cumulative dose. Despite this high rate of exposure to TCE and the rate of complete sequencing (100% for 26 of 48 cases of RCC), only two of the patients for which a mutation was identified had been exposed to TCE and only one of them had been highly exposed the cumulative exposure reaching 830 ppm× years. However this patient had also been exposed to many other occupational risks including exposures to carcinogens (asbestos and ionizing radiation). Three of the patient for which a mutation was identified had been exposed to cutting oils.