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NDT Advance Access originally published online on April 16, 2007
Nephrology Dialysis Transplantation 2007 22(7):2052-2055; doi:10.1093/ndt/gfm184
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Somatic mutations of the von Hippel–Lindau disease gene in renal carcinomas occurring in patients with long-term dialysis

Hitoshi Inoue1, Norio Nonomura1, Yasuyuki Kojima2, Masahiro Shiba1, Daizo Oka1, Yasuyuki Arai1, Masashi Nakayama1, Hitoshi Takayama1, Kazuo Nishimura1, Hiroshi Mori3 and Akihiko Okuyama1

1Department of Specific Organ Regulation (Urology), Osaka University Graduate School of Medicine, Suita, Japan, 2Department of Urology, Inoue Hospital, Suita, Japan and 3Department of Pathology, Osaka Medical College, Takatsuki, Japan

Correspondence and offprint requests to: Dr Norio Nonomura, Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita-city, Osaka 565-0871, Japan. Email: nono{at}uro.med.osaka-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Background. Renal cell carcinoma (RCC) frequently occurs in patients with long-term dialysis. Long-term dialysis causes distinctive pathological changes in the kidney, which is known as acquired cystic disease of the kidney (ACDK). It is of great interest to know whether RCCs occurring in the dialytic kidneys harbour the same or similar mutations of the von Hippel–Lindau (VHL) gene as conventional dialysis-unrelated clear cell RCCs so often do.

Methods. Renal cancer tissues (eight clear cell, two papillary, one Bellini duct and three of the so-called dialysis-specific renal carcinomas) from 13 patients undergoing long-term dialysis were examined for somatic mutations of the VHL disease gene. By means of laser capture microdissection, cancerous and surrounding non-cancerous renal tissues from dialytic patients were subjected to PCR-based direct sequencing of the VHL gene.

Results. Direct forward and reverse sequencing showed that three tumours possessed VHL gene mutations (713delG, 500-504del5-bp and 709A > G). These three mutations were identified in clear cell carcinomas occurring in association with end-stage renal disease undergoing dialysis for 194, 147 and 125 months. None of the non-tumour tissues or other carcinoma tissues analysed, including dialysis-specific carcinoma, possessed VHL gene mutations.

Conclusion. These results indicate that VHL tumour-suppressor gene mutation is involved in clear cell carcinoma in association with long-term dialysis. Mutation of the VHL gene was not found in any of the dialysis-specific RCCs studied herein.

Keywords: acquired cystic disease of the kidney; dialysis; end-stage renal disease; renal cell carcinoma; von Hippel–Lindau



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Renal cell carcinoma (RCC) is the most common malignant tumour of the adult human kidney. The frequency of somatic mutation of the von Hippel–Lindau (VHL) disease tumour-suppressor gene, which is responsible for the VHL familial tumour syndrome, is reported to be ~50% in sporadic clear cell RCCs [1], which account for ~80% of adult sporadic RCCs.

More than 237 000 patients in Japan undergo maintenance dialysis. Among them, 56 000 undergo long-term dialysis (>10 years) because few renal transplants are performed in Japan [2].

RCC occurs at a higher rate in patients with end-stage renal disease (ESRD) than in the general population [3–6] and dialysis of long duration is associated with an increased risk of kidney cancer [5,6]. Most ESRD-associated RCCs are clear cell carcinomas or papillary carcinomas. Clear cell carcinoma is reported to be the most common type of RCC in dialysis patients. According to a questionnaire-based survey in Japan [6], 476 RCCs were diagnosed and reported in total during the period from March 2000 to February 2002, 80.7% (384 cases) of which were estimated as the so-called dialysis-specific RCCs. Among the reported 476 cases, 359 were histologically confirmed to be RCC consisting of 202 clear cell carcinomas, 65 granular cell carcinomas and 52 papillary carcinomas.

Acquired cystic disease of the kidney (ACDK) develops in up to 90% of patients undergoing long-term dialysis [7], with the cysts developing from all tubular segments through continuous proliferation of tubular epithelial cells [8]. The pathology of ACDK appears to involve sequential alterations from tubular epithelium to cyst lining cells, hyperplastic epithelium, adenomaous changes and finally, RCC [9,10].

Proliferative epithelial cells lining cystic cavities in ACDK often exhibit features of cancer cells. These cells with large, frequently mitotic nuclei may form a tumour mass, and this kind of tumour is termed dialysis-specific RCC. According to the General Rules for Clinical and Pathological Studies on RCC supported by the Japanese Urological Association, the Japanese Society of Pathology, and the Japan Radiological Society [11], dialysis-specific RCCs associated with ACDK are classified independently. This type of carcinoma, which was found in 80.7% of 359 hisologically confirmed RCCs in the above-mentioned survey in Japan [6], has not been examined for mutations of the VHL gene so far. The aim of the present study was to determine the frequency of VHL gene mutation in various types of renal carcinomas, including dialysis-specific carcinoma, and their corresponding normal kidney tissues in patients receiving maintenance dialysis.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgement
 References
 
Samples
A total of 14 renal tumour and 13 normal kidney samples were obtained from 13 Japanese patients undergoing maintenance dialysis at the Osaka University Hospital or the Inoue Hospital (Osaka, Japan). One kidney with clear cell carcinoma also showed a separate dialysis-specific carcinoma. Written informed consent was given by all patients for this study. The 14 renal cancers consisted of eight clear cell, two papillary, one Bellini duct and three dialysis-specific carcinomas (Table 1). Blocks of formalin-fixed, paraffin-embedded tissue containing tumour tissue and corresponding non-tumour tissue were prepared. None of the patients had a family history of RCC or symptoms of VHL disease.


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  		Table 1. Renal cell carcinoma in patients with haemodialysis and VHL mutations

 
DNA extraction
After examination of histological sections and identification of tumour and normal tissue boundaries, 14 tumour tissues and 13 non-tumour tissues were dissected from the paraffin blocks using the laser capture microdissection technique (Arcturus Engineering, Santa Clara, CA, USA) (Figure 2). Genomic DNA was extracted with a PicoPure DNA Extraction Kit (Takara Bio Inc., Kusatsu, Japan).


Figure 2
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  		Fig. 2. Histopathology of the kidney in case 8. (A) Non-tumour tissue corresponding to the pelvic region, (B) dialysis-specific carcinoma and (C) clear cell carcinoma.

 
Direct sequencing
To screen for mutations of the VHL gene, six sets of PCR primers [12] were used to amplify fragments covering exons 1–3, containing the VHL gene coding region [12–14]. PCR conditions were as follows: 94°C for 5 min, 40 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 60 s, and a final extension at 72°C for 5 min. Five percent dimethyl sulphoxide was included in PCR reactions for exon 1. We purified PCR products using a PCR Purification Kit (Qiagen K. K., Tokyo, Japan). Direct sequencing was analysed repeatedly. For the VHL gene, all nucleotides were numbered according to the human VHL cDNA sequence originally described by Latif et al. [13]. Codon 1 was located at nucleotide 214, the location of the first methionine residue [15].



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgement
 References
 
We examined a total of 14 cancers and 13 non-tumour tissues for mutations of the VHL tumour-suppressor gene and identified mutations of the VHL gene in three tumours. All three mutations were novel VHL intragenic mutations, not previously reported. In case 6, we found a 5-bp deletion from nucleotide (nt) 500 to nt 504 (Table 1). In case 7, a point mutation (G–A) at nt 709 was identified (Table 1). In case 8, we identified a single deletion (G) at nt 713 that resulted in a frameshift at codon 167 of the VHL gene product (Table 1, Figure 1). The VHL mutation was confirmed to be a somatic change because this tumour-related mutation was not detected in the corresponding normal kidney sample (Figure 2). In this case, a dialysis-specific carcinoma was also present, which did not show any mutation of the VHL gene. Clinical and histopathological data showed these tumours with VHL gene mutations to be typical clear cell RCCs accompanied by ACDK features in patients who had undergone dialysis for 125, 194 and 147 months, respectively. None of the non-tumour tissues or other carcinoma tissues, including dialysis-specific carcinoma, showed VHL mutations.


Figure 1
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  		Fig. 1. Sequence analysis of the VHL gene in case 8. (A) Non-tumour tissue, (B) dialysis-specific carcinoma and (C) clear cell carcinoma.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgement
 References
 
VHL syndrome is an autosomal-dominant, multisystem (pre)neoplastic disorder genetically linked to a germline mutation of a tumour-suppressor gene (VHL-gene) located on chromosome 3p [12]. The phenotypic expression of this tumourous clinico-pathological condition is quite large, and RCC is a common manifestation of VHL syndrome [16,17]. Tumour development is the result of inactivation or loss of the remaining wild-type allele leading to VHL–/– genotype in susceptible cells of various organs [13]. Molecular analysis of the VHL gene has indicated that VHL germline mutations are associated with hereditary clear cell RCC [18]. Evidence has also been accumulated with respect to the association of somatic mutations of the VHL gene with sporadic clear cell RCC [12,19–21], and restoration of VHL gene function in VHL-deficient (VHL–/–) renal carcinoma cells is sufficient to prevent them from forming tumours in vitro [15]. Thus, the VHL gene is considered to play critical roles in the tumourigenesis of sporadic and inherited clear cell RCCs [1,22]. On the contrary, genetic studies have led to the identification of activating mutations of the tyrosine kinase domain of the c-Met protooncogene on chromosome 7 in some tumours of patients with sporadic papillary renal carcinoma [23,24].

It has been shown that RCCs occur frequently in patients undergoing dialysis, particularly patients with ESRD/ACDK. According to a questionnaire-based survey in Japan, the incidence of RCC is 13.3 times higher in men and 15.8 times higher in women with ESRD/ACDK than in the general population [6]. Prolonged dialysis appears to increase the incidence of RCC; the mean duration of dialysis was 102 months for clear cell carcinoma, 186.9 months for granular cell carcinoma, and 166 months for papillary carcinoma [6]. Clear cell carcinoma is reported to be the most common RCC in dialysis patients. In a previous study, mutation of the VHL tumour-suppressor gene was identified in three of seven cases of clear cell carcinoma occurring in association with ESRD/ACDK [25]. In that study, only one non-tumour tissue among seven cases of clear cell carcinoma was examined. In the current study, we examined both eight clear cell carcinomas and their corresponding normal tissues for mutation of the VHL gene using the laser capture microdissection method. Laser microdissection method is necessary to obtain cancer or non-cancer tissue precisely. Three clear cell carcinomas showed VHL gene mutation, whereas the corresponding non-tumour tissues did not. No other types of renal carcinoma in our study showed VHL mutations, including dialysis-specific carcinoma. We have not done northern blot or genomic PCR to detect intragenic and larger deletions because the DNA extracted from paraffin-embedded tissues were not good enough in quality and quantity for such experiments. However, further investigations of both non-tumour tissues and dialysis-specific renal carcinomas are necessary to clarify the pathogenesis of dialysis-specific RCC in patients with ESRD.



   Acknowledgement
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
References
 
This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 

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Received for publication: 9. 1.07
Accepted in revised form: 9. 3.07


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