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VHL Alterations in Human Clear Cell Renal Cell Carcinoma: Association with Advanced Tumor Stage and a Novel Hot Spot Mutation¹

Hiltrud Brauch², Gregor Weirich, Jürgen Brieger, Damjan Glava, Heinz Rödl, Mariola Eichinger, Matthias Feurer, Eberhardt Weidt, Chutintorn Puranakanitstha, Christine Neuhaus, Sigmund Pomer, Walburgis Brenner, Peter Schirmacher, Stephan Störkel, Michael Rotter, Andrej Maera, Nadja Gugeler and Hans-Joachim Decker

Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, 70376 Stuttgart, Germany [H. B., N. G.]; Laboratory of Molecular Pathology, Institute of Pathology, TUM, 81675 Munich, Germany [G. W., H. R., M. E., M. F., M. R.]; Laboratory of Immunobiology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [G. W.]; Hematology, Third Department of Medicine, University of Mainz, 55131 Mainz, Germany [J. B., E. W., C. P., C. N., H-J. D.]; Laboratory of Molecular Genetics, Institute of Pathology, University of Ljubljana, 1000 Ljubljana, Slovenia [D. G., A. M.]; Department of Urology, University of Heidelberg, 69120 Heidelberg, Germany [S. P.]; Department of Urology [W. B.] and Institute of Pathology [P. S.], University of Mainz, 55131 Mainz, Germany; Institute of Pathology, University of Witten-Herdecke, 42283 Wuppertal, Germany [S. S.]; and Department of Molecular Genetics, Bioscientia Institute, 55218 Ingelheim, Germany [H-J. D.]

	ABSTRACT

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To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT₁, pT₂, and pT₃ CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT₃ CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.

	INTRODUCTION

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CCRCC³ is the most common malignant neoplasm of the kidney and belongs to the few human tumors known to evolve from mutations of a specific gene, the VHL tumor suppressor gene (1) . Originally, VHL was identified in families with VHL disease, a rare hereditary multitumor syndrome (1) . Molecular studies have shown that VHL germ-line mutations are associated with hereditary CCRCCs in VHL disease (2) , and moreover, that somatic VHL mutations account for the majority of the more common sporadic CCRCCs (3) . Together with the loss of the homologous chromosome 3p allele (3p LOH), VHL mutations are rate-limiting events in renal tumorigenesis (3 , 4) . Somatic VHL mutations were identified in 33–57% of CCRCCs from the United States, Europe, and Japan (3 , 5, 6, 7, 8) , and in 20% of another subset of CCRCCs, the VHL gene was methylated (9) . Current knowledge suggests that mutations and transcriptional silencing (3, 4, 5, 6, 7, 8, 9) of VHL in renal epithelial cells cause loss or modulation of cellular functions operated by the wild-type VHL protein (pVHL). The molecular mechanisms by which pVHL modulates the expression of target genes leading to CCRCCs are not well understood. Accumulated evidence suggests that pVHL is involved in targeted protein degradation and control of angiogenesis (10, 11, 12, 13) , and there is evidence that pVHL is implicated in regulation of extracellular pH (14) , formation of extracellular matrix (15) , and cell cycle control (10) .

Molecular analyses revealed a broad spectrum of somatic VHL defects. Any nucleotide of the coding sequence downstream of a NotI restriction site may be affected by substitutions, deletions, or insertions (3 , 5, 6, 7, 8) , and other changes may involve methylation (9) . Although most of these alterations seem distributed at random, some occur more frequently. For example, somatic mutations in RCCs cluster within VHL exon 2 (3) . Also, RCCs of patients with defined occupational exposure to a human carcinogen, i.e., trichloroethylene, show frequent cytosine to thymine (CT) transitions and/or a VHL hot spot mutation in exon 1 (16) . With the exception of patients with known environmental exposure for which nonrandom VHL mutations may be linked to a specific carcinogen, the origin of frequent mutations at other sites remains elusive.

Nonrandom distribution of somatic VHL mutations may not only originate endogenously but also from exposure to exogenous carcinogens, which is supported by the observation of regional variations in RCC incidence. For example, for yet unknown reasons, the Bas Rhin region of France has one of the highest incidences of RCC in the world (17) . Furthermore, on the molecular level, the findings of a wide spectrum of somatic VHL mutation frequencies in patients from the United States, Europe, and Japan are unexplained. We reasoned that comprehensive molecular and histopathological data analyses of patients from potentially high-risk areas may provide further clues to the etiology of RCCs and the meaning of somatic VHL alterations.

	MATERIALS AND METHODS

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Renal Tissue and Histopathological Classification.
Normal and tumor tissue of 227 patients with renal epithelial tumors were obtained from three medical centers in Germany. Tumors were designated according to the medical center of origin, i.e., MRI refers to the Institute of Pathology, Technical University Munich-Klinikum rechts der Isar, KT and KTCL to the University Hospital and German Cancer Research Center Heidelberg, and MZ to the University Hospital Mainz. Patients had no history of hereditary VHL disease. Tissue was stored in liquid nitrogen until DNA isolation. Peripheral blood lymphocytes served as a source of normal DNA from patients without available normal renal tissue. Histopathological evaluation (Table 1) and grading were according to Thoenes et al. (18) and in concordance with the recently established classification by the UICC and the AJCC (19) . Readings were independently confirmed by repeated microscopic evaluation by specifically trained pathologists of participating institutes (G. W., M. R., P. S., and S. S). Tumor-Node-Metastasis stages were established according to the UICC (20) . Detailed information on tumors without VHL alterations are available upon request. Informed consent was given by all patients. Investigations were performed retrospectively and approved by a human investigations committee.

LOH was determined by comparison of normal and tumor DNA at microsatellite loci D3S1038, D3S1530, and D3S1435 according to published procedures (24) . For comprehensive evaluation, results from LOH studies from Brauch et al. (25) were included.

Statistical Analysis.
An association between presence of somatic VHL mutations and the CCRCC phenotype compared with lack of somatic VHL mutations in chromophobe RCCs or oncocytomas was calculated according to Fisher’s exact test (26) . Two-sided Ps were used and were considered statistically significant when <0.05. An association between the presence of a somatic VHL mutation and tumor staging (pT) was calculated by contingency table analysis with ² testing (likelihood ratio). All statistical computations were performed with the software SPSS for Windows Rel. 9.0.1 (SSPS, Inc.).

	RESULTS

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All RCCs with somatic VHL mutations and hypermethylations are listed in Table 2 . Carcinomas are consecutively arranged according to affected nucleotides from the 5' to the 3' direction, respectively. Data include pathological characteristics of 77 RCCs, their VHL alterations, i.e., 64 mutations and 13 hypermethylations, as well as 3p LOH of 56 of these RCCs, respectively (Table 2) .

View larger version (7K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Somatic mutations and hypermethylations of the VHL gene. Summary of data from patients with RCCs from three German medical centers (Munich, Heidelberg, and Mainz). The codons of the VHL gene are shown along the X axis. Exon 1 of the VHL gene encodes amino acids 1–114, exon 2 encodes amino acids 114–155, and exon 3 encodes amino acids 155–213. The numbers of mutations or hypermethylations at a particular codon are shown on the Y axis. VHL aberrations identified in RCCs are drawn as perpendicular bars. Most mutations were identified only once, as indicated by the shortest length bar. The methylation-sensitive codon 58 was affected by hypermethylation and a point mutation in 14 patients (n = 14). Codons 147 of 148 were affected in RCCs of nine patients (n = 9). Other mutations that were identified more than once affected codons 98, 114, 115, 122, 126, 143, 151, 156, 158, and 186.

VHL Mutation Hot Spot.
Nine of 77 (12%) RCCs with VHL alterations had a nucleotide change, insertion, or deletion at a thymine cluster (ATT.TTT) within exon 2 encoding amino acids isoleucine and phenylalanine at positions 147 and 148 of pVHL (Table 3) . Examples of deletion T and insertion T are given in Fig. 2 , respectively. In all cases, the mutation was predicted to truncate pVHL at codon 147 or 148.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Mutations in CCRCCs at a mutation hot spot sequence (ATT.TTT) in VHL exon 2. Sequences of CCRCC KTCL 26A, MRI 5, and MRI 31 are compared with the wild-type sequence (wt) in forward (left) and reverse (right) direction. The cell line KTCL 26A shows a deletion T; the sequence is homozygous because of a loss of the homozygous allele and absence of contaminating wild type. MRI 5, a primary tumor, shows the heterozygous sequence pattern of a wild-type allele and a mutated allele with a deletion T. Likewise MRI 31, a primary tumor, shows the heterozygous sequence pattern of a wild-type allele and a mutated allele with an insertion T.

	DISCUSSION

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Similarly to previous studies from the United States, Great Britain, Europe, and Japan (3 , 5, 6, 7, 8, 9) , we established somatic VHL mutations and hypermethylations in 45% of sporadic CCRCCs; however, the observation of a somatic VHL mutation hot spot and the association of VHL mutations with pT₃ CCRCCs are novel observations. Altogether, we observed 60 different genetic and epigenetic changes at VHL, yet 30% of all alterations affected two major sites: (a) 18% of VHL-defective carcinomas showed hypermethylation and frameshift mutation at the NotI restriction site of exon 1; and (b) 12% had somatic VHL mutations that clustered within the nucleotide sequence containing a pentamer thymine repeat. Whereas frequent hypermethylation has been reported previously (9) , only this study revealed frequent somatic mutations at a specific sequence. The independent origin of mutations at the proposed somatic VHL mutation hot spot is suggested by the findings of different mutations, i.e., deletion of adenine, deletion or insertion of a single thymine, and deletions of 5, 7, or 16 nucleotides, and confirmed by independent repetition of experiments. The combined data set of published somatic VHL mutations failed to identify this hot spot (3 , 5, 6, 7, 8) . We propose that our findings of a somatic VHL mutation hot spot point to geographical and/or epidemiological differences in the occurrence of RCCs and CCRCCs, respectively, which is in agreement with the observed high incidence of RCCs in Europe, in particular the Bas Rhin region (17 , 28) .

All mutations at the observed VHL exon 2 mutation hot spot predict truncation of the pVHL at amino acids isoleucine at codon 147 or phenylalanine at codon 148. These residues are located within the S7 unit of the ß sheet domain, and truncation will result in loss of the entire pVHL -domain that contains the critical residues for ElonginC binding (29 , 30) . The structural similarity of ElonginC-VHL with the Skp1-F-box protein complex of the Skp1-Cul1-F-box protein multiprotein complex (which targets proteins for degradation) supports the role of pVHL in an analogous pathway (29) . Thus, tumorigenesis of CCRCCs may be determined by loss of that particular function. Among mutations identified in the germ-line of patients with VHL disease, mutations are biased to occur within the nucleotide sequence encoding the ElonginC binding region. In contrast, somatic mutations mainly seem to affect this region indirectly by mutations at sites 5' upstream within exon 2. The different mutation spectrum between the germ-line and somatic cells was assumed previously to reflect environmental carcinogenic effects (3) . The herein observed frequent thymine cluster mutations within exon 2 support this view. Known mechanisms that may explain thymine mutation involve direct or indirect radiation effects (31) and interaction of DNA with alkylating agents (32) . For example, the formation of cyclobutane pyrimidine dimers is a well-known mechanism to covalently link adjacent pyrimidine rings in the same polynucleotide chain upon UV radiation (33) . The thymine run (ATT.TTT) in VHL exon 2 provides the structural basis of adjacent pyrimidine rings for dimerization to occur. Likewise, alkylating agents known to be electrophilic compounds to interact with strong nucleophilic sites such as N³ -adenine may explain frequent mutations at T-rich sites (34) . Although no defined carcinogen is known to be involved in RCCs of patients of this study panel, shared environmental exposures and life-style as well as variation in individual human susceptibility may contribute to a confounding effect of somatic VHL hot spot mutations.

VHL alterations are considered to be an early event in renal tumorigenesis (3) . Our findings of mutations and hypermethylations in CCRCCs of all stages and nuclear grade are in agreement with the current view of the VHL gene being a gatekeeper in the development of RCCs. Although we were only able to analyze a few small (pT₁) CCRCCs, the presence of VHL mutations in any pT₁ tumor supports the current concept of the VHL gene being an early event in renal tumorigenesis. However, according to another current view, the VHL gene may not be the sole tumor suppressor or gatekeeper gene for CCRCCs and RCCs. This has long been suspected on the basis of LOH data as well as gene mapping efforts and mutation analyses by others (35, 36, 37) . Now, detailed somatic VHL analysis in various tumor stages also supports this view. In light of the current understanding of the pleiotropy of VHL functions (10, 11, 12, 13, 14, 15) , these two concepts are not mutually exclusive but may rather complement each other. Whereas the VHL gene may be a gatekeeper for renal epithelial growth in those RCCs with VHL alterations, those RCCs for which VHL mutations have not been identified may enter the carcinogenic pathway through damage of other genes. Hence, because pVHL participates in numerous cellular control pathways, dysregulation of pVHL without any synchronously underlying VHL gene mutation may be critical in the tumorigenesis of these RCCs. Papillary (chromophilic) RCCs also seem to follow more than one molecular pathway, i.e., mutations in the MET proto-oncogene (36) are responsible for the expression of a distinct morphological phenotype referred to as papillary RCC type 1 (38) . We occasionally observed VHL hypermethylation in highly malignant papillary (chromophilic) RCCs. Our findings are in contrast to a previous report that excluded VHL mutations from RCCs other than nonpapillary (8) , a previously used synonym for CCRCCs.

VHL mutations and hypermethylations seem not to influence malignant behavior determined by nuclear grade but rather may provide a growth advantage to mutated renal epithelial cells in their transition to CCRCCs. We predominantly observed VHL mutations and hypermethylations in stage pT₃ CCRCCs, which invade adrenal gland and perinephric tissue and grow beyond major veins, but we observed fewer mutations and hypermethylations in stage pT₂ tumors limited to the kidney parenchyma. To our knowledge, this is first evidence for a possible link between somatic VHL mutations and a standard prognostic factor. In contrast, CCRCCs associated with germ-line VHL mutations tend to be more indolent than their somatic counterparts (39) . Growth delay in germ-line VHL-affected renal epithelial cells and growth advantage in somatically VHL-affected renal epithelial cells may point to a confounding role of differentiation during tumorigenesis of CCRCCs.

Further evidence for an association of somatic VHL mutations with nonfavorable prognosis comes from the observations of the lack of VHL mutations in chromophobe RCCs and renal oncocytomas. Renal oncocytomas are benign, and the more favorable prognosis of chromophobe RCC has been reported in the literature (40 , 41) . Lack of VHL alterations in these two classes of renal tumors may be a molecular hint for benign or less aggressive behavior. These findings are in agreement with the evolutionary model of renal tumors, according to which CCRCCs and papillary (chromophilic) RCCs develop from proximal tubular cells, whereas chromophobe RCCs and renal oncocytomas derive from intercalated cells of the cortical portion of the collecting duct (18) . Our data suggest that aside from different morphological and biological characteristics, proximal and distal nephrons differ with respect to their susceptibility to acquire somatic VHL mutations. It follows that VHL mutations may be useful in the assessment of RCCs with aggressive biological behavior. The late recognition of chromophobe RCCs as a separate entity among RCCs with "light cell" appearance reflects the difficulty for inexperienced viewers to distinguish less favorable CCRCCs and more favorable chromophobe RCCs (18) . However, the different biological behavior noticed by others makes this distinction clinically important (40 , 41) . We infer that identification of somatic VHL mutation in any unknown "light cell" RCC suggests the diagnosis of CCRCC with less favorable prognosis, which consequently mandates stringent clinical management.

Finally, our work provides another important novel aspect. We noticed the wide range of somatic VHL mutation detection rates reported by others previously, which ranged from 33 to 57% (3 , 5, 6, 7, 8) . Together with hypermethylations (9) , somatic VHL alterations accounted for up to 80% in CCRCCs. Although a 100% VHL mutation detection frequency was reported for germ-line mutations (23) , no study of sporadic tumors showed alterations of all tumors; however, it was inferred that all sporadic CCRCCs may carry somatic VHL alterations. In our study, which technically matches those of other somatic VHL mutation and hypermethylation studies (3 , 5, 6, 7, 8, 9) , we now demonstrated that there may be true differences in somatic VHL mutation detection rates, reflecting the number of pT₃ tumors included in the analyzed patient samples. In these previous studies, no attention was paid to detailed histopathological parameters (3 , 5, 6, 7, 8, 9) . This study showed that the VHL mutation/hypermethylation detection rate was highest in those patient groups with the highest numbers of pT₃ CCRCCs. In contrast, it was lowest in the patient group with predominant pT₂ tumors. These coincidences provided us with the opportunity to establish various VHL mutation/hypermethylation detection frequencies in relationship to tumor staging. The interpretations of our data are based on the sensitivity of enzymatic NotI digestion and Southern blotting as well as SSCP and sequencing to detect most somatic VHL alterations. Our data support the notion that the presence of VHL defects may not only provide a growth advantage to affected CCRCCs but also may allow this growth advantage to be determined at all tumor stages.

ACKNOWLEDGMENTS
We acknowledge establishment of RCC cell lines (KTCL) by D. Komitowski and H. Löhrke, German Cancer Research Center, Heidelberg, and preparation of Figs. 1 and 2 by B. Borstel, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Grants 93.004.1-3 and 93.038.2 from Wilhelm Sander-Stiftung, Grant De356/3-3/4 from the Deutsche Forschungsgemeinschaft, Grant 519, C2 from SFB, and a grant from the Bioscientia Institute Ltd. D. G. was supported by Deutscher Akademischer Austauschdienst and Deutsche Forschungsgemeinschaft, and C. P. was supported by Deutscher Akademischer Austauschdienst.

² To whom requests for reprints should be addressed, at Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Auerbachstrasse 112, 70376 Stuttgart, Germany. Phone: 49-711-8101-3705; Fax: 49-711-85-92-95; E-mail: hiltrud.brauch{at}ikp-stuttgart.de

³ The abbreviations used are: CCRCC, clear cell renal cell carcinoma; RCC, renal cell carcinoma; UICC, Union Internationale Contre le Cancer; AJCC, American Joint Committee on Cancer; VHL, von Hippel-Lindau; pVHL, VHL protein; SSCP, single-strand conformation polymorphism; LOH, loss of heterozygosity; MRI, tumors from University Hospital Munich rechts der Isar, Technical University Munich; KT and KTCL, kidney tumors and kidney tumor cell lines from University Hospital Heidelberg; MZ, tumors from University Hospital Mainz.

Received 8/24/99. Accepted 2/ 1/00.

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