Affiliations of authors: M. Yao, M. Yoshida, T. Kishida, N. Nakaigawa, M. Baba, K. Kobayashi, Y. Kubota, K. Kondo (Department of Urology), Y. Nagashima (Department of Pathology), Yokohama City University School of Medicine, Yokohama, Japan; T. Miura, Department of Urology, Kanagawa Cancer Center, Yokohama; M. Moriyama, Department of Urology, Yokohama City Municipal Hospital, Yokohama; Y. Nakatani, Department of Anatomical and Surgical Pathology, Yokohama City University Hospital, Yokohama.
Correspondence to: Masahiro Yao, M.D., Ph.D., Department of Urology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004 Japan (e-mail: masayao{at}med.yokohama-cu.ac.jp).
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ABSTRACT |
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INTRODUCTION |
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RCC is a morphologically and genetically heterogeneous tumor and can be classified into at least four subtypes, including clear-cell, papillary, chromophobe, and collecting duct (Bellini duct) carcinomas (4,5). The clear-cell subtype accounts for about 80% of all RCCs (5). Intragenic mutation and aberrant hypermethylation of the von HippelLindau disease tumor suppressor gene (VHL) have been found in up to 57% and 19%, respectively, of sporadic clear-cell RCCs (68). In addition, loss of heterozygosity of the VHL gene locus at chromosome 3p25 has been detected in more than 90% of clear-cell RCCs (6,9). These gene alterations did not vary according to tumor progression, including tumor size, stage, grading, distant metastasis, and lymph node metastasis. Thus, inactivation of the VHL gene is thought to be an early or a first event in the clear-cell tumorigenic pathway (10). Reintroduction of the wild-type VHL gene product (pVHL) into VHL-deficient RCC cells can suppress tumor growth in vivo and in vitro (11,12). The VHL gene, therefore, appears to play a critical role in the tumorigenesis of clear-cell RCC. pVHL binds to elongin C/B, Cul2, and Rbx-1 proteins, and this complex targets various proteins, including hypoxia-inducible factor 1 and 2
for ubiquitin-dependent proteolysis (1315). Consequently, pVHL controls the hypoxia-inducible genes, including the genes for vascular endothelial growth factor, the glucose transporter GLUT-1, and carbonic anhydrases (16,17). In addition, VBP1, protein kinase C, Sp1, and fibronectin interact with pVHL, suggesting that the VHL gene may have multiple tumor suppressor functions (1821).
Somatic mutation of the VHL gene is among the most frequent genetic alterations observed in clear-cell RCCs. However, the clinical usefulness of the relationship between VHL mutations and patient survival in sporadic clear-cell RCC (i.e., the prognostic utility of VHL mutations) has not been examined. The purpose of this study was to explore the association between VHL mutations and the risk of death from clear-cell RCC in a large group of patients with long-term follow-up by the use of univariate and multivariable analyses.
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PATIENTS AND METHODS |
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Tumors and corresponding normal kidney samples were collected from nonselected patients who underwent nephrectomy at Yokohama City University Hospital and its affiliated hospitals in the Kanagawa area, Japan, from October 1986 through December 1995. All specimens were frozen rapidly with liquid nitrogen and stored at 80 °C until nucleic acid extraction. All patients were confirmed to have sporadic disease, according to their medical records. Patients with a familial history of RCC or with manifestations associated with VHL disease including retinal angiomas, central nervous system hemangioblastomas, pheochromocytomas, and pancreatic tumors were excluded from the present analysis. The histopathology of the tumors was classified according to the classifications recommended by the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC) (5). Tumor stage and grade were determined after surgical treatment, according to the tumornodemetastasis (TNM) classification (22). We then consecutively analyzed a total of 240 primary renal tumors, including 202 (84%) clear-cell and 38 (16%) non-clear-cell tumors, for somatic VHL alterations by DNAsingle-strand conformation polymorphism (DNASSCP) analysis followed by direct sequencing, Southern blot analysis, and methylation-specific polymerase chain reaction (PCR) assay (23). We found 104 (51%) clear-cell RCCs that showed VHL intragenic mutations. In addition, hypermethylation of the 5' promoter region of the VHL gene was detected in 11 (5.4%) clear-cell RCCs by methylation-specific PCR assay. No VHL alteration was found in any of the 38 non-clear-cell tumors examined. Detailed mutational profiles of these tumors have been described elsewhere (10).
Because the somatic VHL alterations were found exclusively in clear-cell tumors, all subsequent analyses were restricted to those patients with clear-cell RCC. Among 202 patients with clear-cell RCC, 15 (7.4%) were excluded from prognostic assessment because of inadequate follow-up (13 were lost to follow-up, and two lacked adequate clinical information). The remaining 187 (93%) patients were finally included in the present analysis. The study protocol was approved by the institutional ethics committee. One hundred thirty-four patients (72%) with stage IIII tumors (UICC classification) underwent potentially curative nephrectomy, whereas the remaining 53 patients with stage IV disease received a palliative or adjunctive nephrectomy. Patients did not receive any preoperative therapy. Ninety-four (70%) of the 134 patients with a stage IIII tumor received postoperative interferon (IFN-
) and/or chemotherapy as an adjuvant treatment. The majority of the patients (88 of a total of 94 patients; 94%) who received an adjuvant treatment had relatively large tumors (
5 cm), higher stage tumors (stage II or higher), or higher grade tumors (grade 2 or higher). Clinicopathologic data, including sex distribution, age, symptomatic presentation, UICC stage, and tumor grade, are listed in Table 1
. All patients were followed up by urologists at intervals of 16 months. Follow-up was 65.1 ± 38.9 months (mean ± standard deviation; range = 2155 months) after surgical treatment. As of October 2001 when follow-up ended, 110 patients were alive without disease, 13 had died without disease recurrence, nine were alive with disease recurrence, and 65 had died of the disease.
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The 2 test was used to test for differences in distribution between groups. Survival time was defined as the time from nephrectomy until the patients death, known recurrence, or the last time that the patient was known to be alive. Both cancer-free survival and cancer-specific survival were estimated by the KaplanMeier method, and the resulting curves were compared by use of the log-rank test. The Cox proportional hazards model with backward selection procedures was used to examine the simultaneous effects of several variables on patients outcome. The data were consistent with the assumptions of Coxs proportional modeling. All statistical analyses were performed with SPSS software (version 10.1; SPSS, Inc., Chicago, IL). All statistical tests were two-sided and were considered to be statistically significant at P<.05.
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RESULTS |
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The VHL gene mutational status for 187 clear-cell RCCs was determined by DNASSCP followed by direct sequencing, Southern blot analysis, and methylation-specific PCR assays. A VHL alteration was detected in 108 (58%) samples: 98 samples with intragenic mutations (46 deletions, 30 missense mutations, 16 insertions, and six nonsense mutations) and 10 with hypermethylation. We did not find any aberrant Southern blot patterns in the tumors examined. VHL mutations and their relation to various clinicopathologic parameters are listed in Table 1. VHL alteration was statistically significantly less common among younger patients (
55 years) with clear-cell RCC (
2 P = .013). However, no statistically significant association was found between a VHL mutation and any of the other clinicopathologic parameters, including patient sex, symptom presentation, tumor stage, and tumor grade (Table 1
). It should be noted that, in patients with stage IIII tumors treated by radical nephrectomy, there was no difference in the VHL mutational detection rate between those receiving postoperative adjuvant therapy and those not receiving adjuvant therapy (
2 P = .623). Because the postoperative treatment for each patient was determined according to clinicopathologic status and patient status and because the VHL mutation analysis was performed at least several months after the surgical resection, we believe that there was no treatment or follow-up bias between patients with VHL mutations and patients without VHL mutations.
VHL Alteration and Patient Survival Time
We first examined VHL mutational status and survival of the patients with clear-cell RCC by focusing on the 134 potentially curable patients with UICC stage IIII tumors who received a radical nephrectomy. This is because complete surgical resection, defined by the absence of macroscopic or microscopic remnant disease, is considered to be the only treatment for which a complete cure for patients suffering from RCC can reasonably be expected. In the univariate analyses, cancer-free survival and cancer-specific survival were strongly associated with symptomatic presentation, tumor stage, tumor grade, and VHL mutational status (Table 2). KaplanMeier survival analysis demonstrated that the presence of VHL alteration was associated with better cancer-free survival (P = .024) and cancer-specific survival (P = .023) (Fig. 1
). We further explored the prognostic value of VHL alteration in a stage-specific manner. A statistically significant association between VHL alteration and better outcomes in terms of cancer-free survival and cancer-specific survival was observed in patients with relatively advanced disease (stage III [P = .014 and .010, respectively] or stage II + III [P = .002 and .009, respectively]) (Table 3
). In addition, similar associations were found in patients with relatively higher grade tumors (grades 3 and 4 [P = .013 and .032] or grades 2, 3, and 4 [P = .013 and .018, respectively]) and in patients with symptoms at presentation (P = .005 and .012) (Table 3
).
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We next examined VHL mutational status and survival of the 53 patients with stage IV tumors who received a palliative or adjunctive nephrectomy. We found that 30 patients had VHL alterations in their tumors and 23 did not. In each of these groups, 28 (93%) of the 30 patients and 21 (91%) of the 23 patients were treated postoperatively with IFN- alone or with a combination of IFN-
and chemotherapeutic agents, such as 5-fluorouracil and/or vinblastine. A KaplanMeier survival analysis demonstrated that the VHL mutational status had no influence on cancer-specific survival rates for patients with stage IV tumors (log-rank P = .760).
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DISCUSSION |
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In this study, we have shown that the presence of VHL alteration (mutation or hypermethylation) is associated with better outcomes for patients with stage IIII clear-cell RCCs treated by potentially curative nephrectomy. Of particular interest, these associations were more statistically significant for patients with a relatively high risk for tumor recurrence or death, including higher stage, higher grade, and symptoms at presentation. To our knowledge, this is the first report of an association between VHL alteration and survival in patients with sporadic clear-cell RCC. Our data suggest that VHL alteration status may provide useful prognostic information, as a biomolecular marker, for patients with clear-cell RCC.
The VHL gene was originally isolated and identified as a tumor suppressor gene responsible for von HippelLindau familial tumor syndrome predisposing to the development of retinal angiomas, central nervous system hemangioblastomas, pancreatic tumors, pheochromocytomas, and multiple, bilateral clear-cell RCCs (24). Mutational analyses have demonstrated that nearly 100% of VHL kindreds have germline VHL alterations, such as intragenic mutations, Southern blot aberrations, and large genomic deletions (2527). Moreover, somatic intragenic mutations have been found in 42%57% of sporadic clear-cell RCCs (6,7,9,28). Aberrant hypermethylation of the VHL promoter region has been detected in an additional 5%19% of these tumors (8,9,29). Reintroduction of the wild-type pVHL into VHL-deficient RCC cells can suppress tumor growth (11,12). Thus, the VHL gene appears to have a critical function in the tumorigenesis of clear-cell RCC. In contrast to germline mutations, 30%40% of sporadic clear-cell RCCs did not have a somatic or epigenetic VHL change, suggesting that the VHL-independent tumorigenic pathway may be involved in the subset of clear-cell RCCs (30). Our data clearly demonstrate that RCCs without a VHL alteration have a poorer prognosis than RCCs with a VHL alteration. Around 40% of clear-cell RCCs may have a VHL-independent tumorigenic pathway and, consequently, express a different biologic behavior than clear-cell RCCs with a VHL alteration. Previous studies suggested that the clear-cell RCCs in VHL kindreds tend to grow slowly and that patients with such tumors have better outcomes than those with sporadic RCC (31,32). Our data appear to support these observations. RCCs in VHL kindreds invariably have a VHL-dependent tumorigenic pathway. Therefore, as demonstrated in our study, patients with RCCs in VHL kindreds are likely to have better outcomes than those with sporadic RCC.
We examined a possible association between patient survival and VHL mutational subtype (missense versus nonmissense mutations) because previous studies indicated that nonmissense VHL mutations are predominantly associated with sporadic and germline cases of clear-cell RCC (28,33). Missense mutations generally change only one amino acid in the whole pVHL, but nonmissense mutations are likely to grossly disrupt pVHL function or completely block the expression of pVHL. In our patient series, we did not detect any survival difference between these two mutational groups, although the sample size was relatively small (21 with missense mutations and 57 with non-missense mutations, including 34 deletions, 12 insertions, five nonsense mutations, and six hypermethylations). Thus, the type of VHL mutation in clear-cell RCCs does not appear to influence tumor progression, invasion, or metastatic processes. These mutations may instead be involved in the initial malignant transformation of renal tubular cells, as previously predicted (6,10,34).
We did not find any survival difference between patients with stage IV tumors treated by a palliative or adjunctive nephrectomy who did or did not have altered VHL genes. The majority of these patients were treated with IFN- alone or IFN-
combined with chemotherapeutic agents after nephrectomy. Clinical data demonstrated that the response rate to IFN-
-based treatment in patients with advanced RCC was 10%20% (35). From our data, it appears difficult to predict the response of advanced or metastatic RCC to treatment with IFN-
solely on the basis of VHL mutational status. However, our patient series was again limited and relatively small (30 patients with a VHL alteration and 23 patients without a VHL alteration). Prospective studies with a larger number of patients may be needed to clarify these issues.
In summary, we have demonstrated a strong association between VHL gene alteration and patient outcome, particularly in patients with stage IIII clear-cell RCC who undergo radical nephrectomy. The presence of a VHL gene alteration, therefore, may be useful as a prognostic biomarker in such patients. Our data also indicate that clear-cell RCCs with and without VHL alterations show different biologic behaviors. Although morphologic differences in these tumor subtypes have not yet been distinguished, molecular genetic approaches based on VHL mutation or, alternatively, molecules located downstream of the VHL gene may be applicable for a novel diagnosis of such tumors.
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NOTES |
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We thank Yoko Nakamura, Miyuki Ishii, and Rie Shimizu for their excellent technical assistance.
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Manuscript received January 30, 2002; revised June 17, 2002; accepted August 8, 2002.
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