Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
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Clinical Cancer Research 13, 152-160, January 1, 2007. doi: 10.1158/1078-0432.CCR-06-1877
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Imaging, Diagnosis, Prognosis

Expression of Adipose Differentiation-Related Protein: A Predictor of Cancer-Specific Survival in Clear Cell Renal Carcinoma

Masahiro Yao1, Ying Huang1, Koichi Shioi1, Keiko Hattori1, Takayuki Murakami1, Noboru Nakaigawa1, Takeshi Kishida1, Yoji Nagashima2 and Yoshinobu Kubota1

Authors' Affiliations: Departments of 1 Urology and Molecular Genetics and 2 Molecular Pathology and Oncology, Yokohama City University Graduate School of Medicine, Yokohama, Japan

Requests for reprints: Masahiro Yao, Department of Urology and Molecular Genetics, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. Phone: 81-45-787-2679; Fax: 81-45-786-5775; E-mail: masayao{at}med.yokohama-cu.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We recently found that adipose differentiation-related protein (ADFP) is a potential diagnostic and prognostic biomarker for clear cell subtype renal cell carcinoma (RCC). To further evaluate the correlation between ADFP expression levels and clinicopathologic characteristics and patient outcome, we retrospectively examined patients with clear cell RCC.

Experimental Design: A series of 432 consecutive patients with sporadic clear cell RCC who underwent nephrectomy between March 1986 and June 2004 were enrolled in the study. ADFP expression levels in the primary tumors and in 18 metastases were measured by real-time quantitative PCR. The clinicopathologic and prognostic data were collected, as well as the von Hippel-Lindau disease (VHL) gene alteration status in selected cases.

Results: ADFP expression was apparently high in cases without a symptomatic presentation, as well as in cases of low-stage, low-grade, or VHL alteration–positive clear cell RCC, whereas it was down-regulated in undifferentiated tumors with a spindle/pleomorphic component or metastatic lesions. Univariate analyses showed that high ADFP expression was associated with better cancer-specific survival and cancer-free survival. Further Cox multivariate analyses combined with the split-sample validation method showed that ADFP expression still remains an independent predictor for cancer-specific survival in all tumor stages and in advanced metastatic cases, whereas the predictive value of ADFP expression for cancer recurrence is rather weak.

Conclusions: The ADFP expression may represent the tumor differentiation status, and the detection of the expression levels provides useful prognostic information for cancer-specific survival in patients with clear cell RCC.

Renal cell carcinoma (RCC) is the most common malignant tumor in the adult kidney, accounting for ~3% of human malignancies (1). The occurrence of RCC has increased in recent years; worldwide, almost 100,000 patients die of the disease every year (2). Complete surgical resection is considered to be the only effective treatment for patients with clinically localized RCC. However, the disease will recur postoperatively in 20% to 40% of patients who undergo potentially curative nephrectomy (1, 2). Although various therapeutic strategies, including combined chemotherapies and/or immunotherapy by cytokines, have been applied to the disease, the prognosis for RCC patients with metastases is quite poor (1, 2). To improve the survival rate of RCC patients, there is a desperate need to develop novel therapeutic agents for selective molecular targets, as well as useful tumor markers, for early detection of the disease and for the accurate prediction of patient outcome (3, 4).

Recent studies have clearly shown that RCC is a morphologically and genetically heterogeneous tumor and can be classified into at least four major subtypes: clear cell, papillary (chromophilic), chromophobe, and collecting duct carcinomas (5, 6). Among them, the clear cell tumor subtype is the most frequent, accounting for ~80% of all RCCs (5, 6). Regarding its molecular pathogenesis, loss of the von Hippel-Lindau (VHL) tumor suppressor function, due to somatic mutation, loss of heterozygosity, and hypermethylation of the promoter region, is frequently observed (7, 8). Further functional studies have shown that the VHL gene plays a crucial "gatekeeper" role in the tumorigenesis of clear cell RCC (4, 9, 10).

By means of microarray gene expression analysis, we have searched for potential biomarkers for diagnosing renal tumor subtypes and predicting prognosis. As a result, we identified >100 genes specifically overexpressed in clear cell subtype RCC. Among them, we found that adipose differentiation-related protein (ADFP) is highly up-regulated both at the transcriptional and protein levels in clear cell RCC cells (11). ADFP is characterized as one of the crucial proteins involved in fatty acid uptake and in the formation and stabilization of lipid storage droplets (12, 13). ADFP is expressed at high levels in adipocytes and is also expressed at various levels in many different types of cells, including lactating mammary epithelial cells, adrenal cortex cells, Sertoli and Leydig cells of the male reproductive system, and fatty change hepatocytes in alcoholic liver cirrhosis (14–17). Of interest, ADFP is characterized as one of the hypoxia-inducible genes, and its transcriptional activation is mediated by hypoxia-inducible factor (HIF; ref. 18). It has been shown that HIF-{alpha} is the critical target for ubiquitin-dependent proteasome degradation by the VHL-ElonginBC-Cul2/Rbx ubiquitin ligase complex in clear cell RCC cells (4, 9, 10). Collectively, these findings suggest that disruption of the VHL tumor suppressor/HIF pathway is likely involved in the up-regulation of ADFP in clear cell RCC (11). Moreover, our preliminary data suggested that patients with tumors that showed a high level of ADFP expression had better survival rates than patients with tumors having a low level of ADFP expression (11).

In the present study, to further elucidate the significance of ADFP as a molecular expression marker, we examined 432 consecutive patients with sporadic clear cell RCC. In selected cases, we also measured ADFP expression levels in the metastatic tumor tissues concomitant with the primary RCC and compared these expression levels. We used the Cox regression model combined with the split-sample method (i.e., a training-test approach) to validate the survival tests.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients with clear cell RCC. Primary tumors and corresponding normal kidney samples were collected from 432 consecutive patients with sporadic clear cell RCC who underwent nephrectomy between March 1986 and June 2004. Eighteen metastatic tumor lesions were also resected as a surgical treatment for selected patients. Advanced metastatic patients were basically treated by the standard protocols, mainly by IFN-{alpha}– and/or low-dose interleukin-2–based immunotherapy. None of the patients received novel experimental therapies, such as allogeneic stem cell transplantation, tumor vaccines, or molecular targeted medicines (19, 20). Fresh tumor materials without apparent necrosis were grossly resected, snap-frozen with liquid nitrogen, and stored at –80°C until nucleic acid extraction. The histopathology of the tumors was classified according to standard classifications (5, 6). Tumor stage and grade were determined according to the tumor-node-metastasis classification (21). Among the 432 patients with clear cell RCC, 103 cases had already been examined in a previous report on the association of ADFP with clinicopathologic characteristics (11). We newly studied the ADFP expressions in the remaining 329 cases. Of the 432 patients, presumably curative surgery was done in 339, including 329 Unio Internationale Contra Cancrum stage I to III patients who underwent nephrectomy (313 conventional and 16 partial nephrectomies) and 10 stage IV patients with metastases in a solitary organ who underwent radical nephrectomy together with curative surgical resection for the metastases. Cancer-free survival was therefore determined for this 339-patient cohort. The remaining 93 patients with stage IV disease received a palliative or adjunctive nephrectomy. All patients were followed up by urologists at intervals of 1 to 6 months. As of January 2006, when the follow-up ended, the follow-up times had ranged from 0.4 to 236.5 months and the median follow-up time after nephrectomy was 68.7 months for all patients and 84.5 months for survivors (Supplementary Table S1). The study protocol was approved by the institutional review board.

Measurement of ADFP expression by real-time quantitative PCR. Frozen tissue fragments without microdissection were mechanically disrupted in Isogen reagent (Nippon Gene, Tokyo, Japan) using a microtube homogenizer (Toyobo, Osaka, Japan) and total RNA was immediately isolated. cDNA preparations and real-time quantitative PCR with a TaqMan fluorescent probe for the measurement of ADFP expression were done essentially as previously described (11). In each experiment, at least two independent real-time quantitative PCR reactions were done to obtain the mean expression values. Obtained signals were normalized by dividing by the mean expression signal of ß-actin.

Statistical analysis. The {chi}2 test, independent samples t test, Mann-Whitney U test, Wilcoxon signed-rank test, or Kruskal-Wallis H test was used to examine differences between groups depending on the data set. To define the optimal cutoff value of ADFP expression in the real-time quantitative PCR analysis, as well as the tumor size, we applied the receiver operating characteristic method to estimate candidate cutoff point regions. We then searched for the optimal cutoff point for these characteristics, which showed the maximum statistical power by the Kaplan-Meier cancer-specific survival estimation with the log-rank test (Supplementary Figs. S1 and S2). Survival time was defined as the time from nephrectomy or, in cases with recurrence, the time from when the tumor recurrence was discovered until the patient's death or the last time that the patient was known to be alive. Both cancer-specific survival and cancer-free survival curves were estimated by the Kaplan-Meier method, and the resulting curves were compared using the log-rank test. Univariate and multivariate analyses were done using the Cox regression models. In multivariate analysis, the Cox proportional hazards model was used to examine the simultaneous effects of several variables on patients' outcomes. All data were consistent with the assumptions of Cox proportional modeling. All statistical analyses were done with SPSS software (SPSS, Inc., Chicago, IL). All statistical tests were two sided with P < 0.05 considered as statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
ADFP expression levels, clinicopathologic variables, and VHL alteration in clear cell RCC. We examined a total of 432 primary sporadic clear cell RCCs as well as 49 corresponding normal kidney samples for ADFP expression levels using real-time quantitative PCR (the detailed clinicopathologic characteristics are listed in Supplementary Table S1). The median signal values detected in the real-time quantitative PCR for primary tumors (n = 432) and normal kidney tissue samples (n = 49) were 1.319 and 0.429, respectively. Therefore, as previously observed (11), ADFP was clearly up-regulated in clear cell RCCs compared with normal kidney tissues (P = 9.37e–16, Mann-Whitney U test).

We next explored possible correlations between ADFP expression levels and various clinicopathologic characteristics in clear cell RCC cases. The ADFP expression level was significantly higher in cases without a symptomatic presentation, as well as in lower-stage or lower-grade tumors, compared with normal kidney tissue (Table 1 ). ADFP was originally characterized as one of the differentiation markers for adipocytes (12). Among the 432 primary RCCs studied, 40 (9.3%) tumors contained a spindle/pleomorphic histologic component, which is considered to be an undifferentiated or dedifferentiated manifestation of conventional RCC (5, 6, 22). When we fractionated these 40 tumors and measured their ADFP levels, we found that the ADFP expression was significantly lower compared with that of conventional clear cell subtype RCCs with G1 to G3 grading (Fig. 1A ). In addition, among conventional clear cell RCCs, there was a tendency for the level of ADFP expression to decrease with increasing tumor grade (Fig. 1A; Table 2 ). These correlations suggested that in clear cell RCC, ADFP levels are representative of the tumor differentiation status.


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  		Table 1. Correlation between clinicopathologic characteristics and VHL alteration status and ADFP expression level in patients with clear cell RCC

 

Figure 1
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  		Fig. 1. Expression Levels of ADFP by real-time quantitative PCR in the histologic tumor grading (A) and the paired samples with a primary tumor and its metastasis (B). A, the ADFP expressions in each category are shown by the dot plot as well as by the box-whisker plot. The box depicts the borders of 25% and 75% quartiles; horizontal bar, median value. Whiskers, ranges. Nk, normal kidney tissue; Sp/Pl, spindle/pleomorphic. B, the ADFP expression signal values in the pairing of the primary tumor and its metastasis. Prt, primary tumor; Met, metastasis. P values were calculated using Wilcoxon signed-rank test.

 

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  		Table 2. Expression levels of ADFP in the histologic tumor grading and in normal kidney tissues

 
Among the 432 clear cell RCC cases, we previously measured the VHL gene alteration status in 220 cases, and these earlier results showed that 116 (53%) cases had a somatic intragenic mutation or hypermethylation of the VHL gene (8, 23). ADFP expression levels were clearly higher in VHL alteration–positive clear cell RCCs compared with VHL alteration–negative ones (P = 0.010, Mann-Whitney U test; Table 1). ADFP expression did not correlate with the other clinicopathologic variables (i.e., patient age, sex, tumor size, and tumor vascular invasion status; Table 1).

Comparison of the ADFP expression levels in the primary tumors and metastatic lesions. Among our RCC cases, a total of 18 metastatic lesions (6 lung, 4 bone, 4 contralateral kidney, 2 skin, 1 adrenal, and 1 brain metastasis) in 14 patients were surgically resected and available for the real-time quantitative PCR analyses together with the primary RCCs. When the ADFP expression levels were compared between the primary tumors and their paired metastases, the metastatic lesions showed a clearly lower ADFP expression than did the primary tumors overall (P = 0.001, Wilcoxon signed-rank test; Fig. 1B). It is notable that the ADFP expression values for all 18 metastases were appreciably low (median, 0.418; range, 0.049-1.369) and comparable with those of primary tumors with a spindle/pleomorphic component (Fig. 1A and B). Further investigation of the paired samples suggested that there may be two classes categorized according to the ADFP expression patterns: one class containing 8 (44%) pairings in which the primary tumor expressed a relatively high level of ADFP and the metastasis showed down-regulation of ADFP, and another class comprising the remaining 10 (56%) pairings in which the ADFP expression was fairly low both in the primary tumors and the metastases (Fig. 1B).

Survival test models in the split-sample method. We next explored the association between ADFP expression levels and the survival of patients with clear cell RCC. For this purpose, we applied the split-sample method (i.e., a training-test set approach) to validate the survival test (24). We divided the patients into two groups: the preliminary training set consisting of 182 patients treated in the earlier period (March 1986-October 1994) and the validation test set group consisting of the remaining 250 patients who were treated in the later period (November 1994-June 2004). The optimal cutoff value for ADFP expression in the training set group was then determined to be 0.910. Based on this cutoff, 116 of the 182 (64%) clear cell RCCs, of which the ADFP expression levels were ≥0.910, were designated high expression tumors, and the remaining 66 (36%) tumors, of which the ADFP expression was <0.910, were designated low expression tumors. Using the same method, the tumor size was classified into three categories: ≤4.5, 4.6 to 9.4, and 9.5 cm (Supplementary Figs. S1 and S2). We then applied these cutoff values in the following survival analyses.

ADFP expression and survival of patients with clear cell RCC. We examined the ADFP expression levels and survival rates of patients with clear cell RCC. In the univariate analyses, ADFP expression levels were strongly associated with cancer-specific survival for the training set, test set, and all-patient cases (Fig. 2A ; Supplementary Table S2). The patients with tumors that showed high ADFP expression had significantly better cancer-specific survival than the patients with tumors that showed low ADFP expression. On the other hand, in cancer-free survival tests, the statistical significance of ADFP was detected for the test set and the all-patient cohort but not for the training set group (Fig. 2B; Supplementary Table S3). In the present univariate analyses with the split-sample model, as in the previous study, clinicopathologic characteristics such as symptomatic presentation, tumor size, tumor stage, grading, and microvascular invasion status showed a strong association with both cancer-specific survival and cancer-free survival rates (except for the tumor stage in the training set/cancer-free survival; Supplementary Tables S1 and S2).


Figure 2
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  		Fig. 2. Kaplan-Meier survival probability and ADFP expression levels in the training set (A), test set (B), and all patients (C) with clear cell RCC. A, cancer-specific survival of patients with clear cell RCC who underwent nephrectomy. B, cancer-free survival of patients with clear cell RCC who underwent presumably curative surgical resection. C, cancer-specific survival for patients with advanced metastatic clear cell RCC. Stage IV clear cell RCC patients who underwent noncurative surgery (n = 93) and patients in whom metastasis occurred after presumably curative nephrectomy (n = 71) were combined, and the survival probabilities were evaluated as a single cohort.

 
Next, we used multivariate analysis to determine whether the association between ADFP expression and patient survival was independent of other outcome predictors. The Cox proportional hazards model for all characteristics showed that the ADFP expression remained a statistically significant independent predictor for the test set and the all-patient cohort in cancer-specific survival tests (Table 3 ). On the other hand, for cancer-free survival tests, a significant ADFP expression was not detected in any of the patient cohorts tested (Table 4 ).


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  		Table 3. Multivariate analyses of cancer-specific survival among patients with clear cell RCC in the test set and all patients

 

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  		Table 4. Multivariate analyses of cancer-free survival among patients with clear cell RCC in the test set and all patients

 
ADFP is a survival predictor for advanced metastatic clear cell RCC. Finally, we studied the effect of ADFP expression on the survival time of advanced metastatic RCC patients with apparent tumor burdens. A total of 93 patients with stage IV disease received a palliative surgery, and 71 patients who had developed tumor recurrence were combined and enrolled for the analysis. In the univariate analyses, we detected significant survival differences between the ADFP high and low expression groups for the training set, test set, and all-patient cases (Fig. 2C). In further multivariate analysis models examining five variables (patient age, sex, tumor grade, vascular invasion, and ADFP expression), ADFP expression was still found to be an independent predictor of survival time for all three patient cohorts (Table 5 ).


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  		Table 5. Multivariate analyses of cancer-specific survival among patients with advanced metastatic clear cell RCC in the training set, test set, and all patient cases

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We evaluated ADFP expression, a potential prognostic factor, in a relatively large cohort of patients who suffered from clear cell RCC and who had been followed up for a substantial period of time; the cohort contained all tumor stages. We measured ADFP expression levels by means of the TaqMan-based real-time quantitative PCR assay, which is a simple, rapid, and sensitive transcription detection method that is applicable to current clinical practice (25, 26). We applied the Cox regression model combined with the split-sample method to survival tests as a validation method because the study was basically designed in a retrospective manner (24, 27). We found that the RCC patients with high expressions of ADFP showed better outcomes than those with low expressions of ADFP in both cancer-free survival and cancer-specific survival tests. Furthermore, this validation method enabled us to show that ADFP expression is an independent predictor of cancer-specific survival in a patient cohort containing all tumor stages, as well as for a cohort of patients with advanced metastases (i.e., stage IV cases or tumor recurrent cases), and is of special value because only a few informative biomolecular markers have been identified for patients with such tumor burdens (3).

The current study supports our previous finding that ADFP was up-regulated both at the transcriptional and protein levels in cancerous cells of clear cell RCC, whereas ADFP protein existed at levels almost undetectable by immunohistochemistry in normal kidney tissues, including proximal tubular epithelial cells, which are considered to be precursor cells for clear cell subtype RCC (11, 22). We confirmed again that the ADFP message detected by real-time quantitative PCR is clearly high in clear cell RCCs compared with normal kidney tissue samples. Moreover, we showed that among conventional clear cell subtype RCCs, the ADFP expression levels tended to decrease with increasing grade of tumor, and that, consequently, these levels were significantly low in tumors with a spindle/pleomorphic component, the dedifferentiated histology of which indicates an aggressive and highly metastatic potential (5, 6, 22, 28).

In our previous study, ADFP protein was mainly stained in clear cell RCC cells by immunohistochemistry, and the ADFP expression levels quantified by immunohistochemistry and real-time quantitative PCR assay in selected tumors (n = 28) were well correlated ({rho} = 0.601, Spearman's rank correlation test; ref. 11).3 We therefore believe that the ADFP mRNAs detected by the real-time quantitative PCR assay are primarily a reflection of the expression in tumor cells, although we used grossly dissected tissue samples. Alternatively, the immunohistochemistry-based ADFP semiquantification would also be useful for predicting the survival of patients with clear cell RCC.

It is now widely believed that multiple genetic changes accumulate during the sequential processes of cancer transformation, tumor progression, and the acquisition of aggressive and metastatic potential (29, 30). Our findings suggested that after ADFP is transcriptionally activated in a relatively early step, it is gradually down-regulated during the later dedifferentiation processes in the tumorigenesis of clear cell RCC. We observed that the VHL alteration–positive clear cell RCCs expressed higher levels of ADFP compared with the VHL alteration–negative ones. The inactivation of the VHL tumor suppressor is a hallmark of clear cell RCC and is considered to be the initial step in the multistage tumorigenic model (4, 9, 10). The ADFP transcription is known to be mediated by HIF (18). HIF-{alpha} is one of the critical targets for ubiquitin-dependent proteasome degradation by the VHL-ElonginBC-Cul2/Rbx1 ubiquitin ligase complex in clear cell RCC (4, 9, 10).

We had previously examined gene expression profiles both in pVHL-deficient and wild-type pVHL-positive RCC cells of various cell densities by means of the in vitro VHL introduction model (31). We observed that the transcription of ADFP was repressed by the introduction of wild-type pVHL. Its repression levels were comparable with those of vascular endothelial growth factor or glucose transporter-1.3 These are well characterized as major targets for the VHL/HIF signaling cascade in RCC cells (32). Taken together, these findings strongly suggest that the up-regulation of ADFP is caused by the disruption of the VHL/HIF pathway in clear cell RCC.

Very recently, carbonic anhydrase IX (CA9) and hypoxia-inducible protein 2 (HIG2) have been identified as potential diagnostic and prognostic biomarkers for clear cell RCC (33, 34). It is notable that, like ADFP, both molecules are located in the region downstream of the VHL/HIF signaling (34, 35). Moreover, CA9 and HIG2 are expected to have potential applications for therapeutic interventions (34, 36). We previously showed that VHL-mutated clear cell RCCs show better survival rates than VHL-mutation negative RCCs (37). Therefore, these newly characterized prognostic biomarkers are likely to reflect, in part, the VHL mutational status. Conversely, other molecules located downstream of the VHL/HIF cascade could be potential biomarkers and/or even novel therapeutic targets for clear cell RCC.

It is also known that ADFP is transcriptionally activated by the peroxisome proliferator-activated receptor (PPAR)–mediated pathway (38). The PPAR family consists of three isotypes, PPAR{alpha}, PPARß/{delta}, and PPAR{gamma}. These isotypes belong to a subfamily of nuclear receptors that heterodimerize with retinoid X receptors and regulate the transcription of various target genes, including ADFP. PPARs play fundamental roles in regulating energy homeostasis, including lipid and glucose metabolisms (39). Moreover, PPARs, the {gamma} isoform in particular, are involved in the modulation of the growth of cancerous cells in a wide range of organs, such as in carcinomas of the colon, lung, thyroid, bladder, pancreas, and kidney (40–42). PPAR{gamma} is known to be highly expressed in RCC (43). Moreover, treatment of RCC cells with ligands of PPAR{gamma} has been shown to induce growth inhibition and apoptosis (43, 44). In addition to VHL/HIF, the PPAR{gamma}-mediated signaling cascade and/or its activated status may play a role in modulating the ADFP expression status in clear cell RCC.

In the present study, we found that the ADFP expression levels in the metastases were very low and were comparable with those in the spindle/pleomorphic RCC. Interestingly, in cases where the primary tumor showed a relatively high level of ADFP, ADFP was down-regulated in its metastasis, whereas in other cases, the levels of ADFP were fairly low in both the primary tumor and its metastasis. The classic paradigm of metastasis proposes that rare subpopulations within primary tumors acquire metastatic capability via sequential mutations, suggesting that metastases are genetically dissimilar from their primary tumors (30, 45). On the other hand, more recent data suggest that primary tumors have already acquired genetic changes capable of aggressive and metastatic potential. Thus, the molecular genetic profiles of both the primary tumor and its metastatic lesions have been shown to be quite similar (46, 47). The ADFP expression signatures between the primary tumor and its metastasis suggest that both metastatic patterns may exist in clear cell RCC, although the number of analyzed samples was limited.

In summary, we showed that ADFP is a novel prognostic expression signature for clear cell RCC. The measurement of ADFP expression should serve as a useful index of cancer-specific survival rates of patients with all tumor stages as well as those with advanced metastases. In addition, the elucidation of the signal cascades and/or molecules modulating ADFP should help to advance our understanding of the biology of clear cell RCC.


   Footnotes
 
Grant support: Grants-in-Aid for Scientific Research nos. 16591610 and #18591764 from the Ministry of Education, Science, Sports, and Culture of Japan (M. Yao) and the Strategic Research Project nos. 2005-K17017 and 2006-K18029 of Yokohama City University, Japan (M. Yao).

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.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

3 M. Yao, personal communications. Back

Received 7/28/06; revised 9/19/06; accepted 10/ 6/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Motzer RJ, Bander NH, Nanus DM. Renal-cell carcinoma. N Engl J Med 1996;335:865–75.[Free Full Text]
  2. Vogelzang NJ, Stadler WM. Kidney cancer. Lancet 1998;352:1691–6.[CrossRef][Medline]
  3. Lam JS, Shvarts O, Leppert JT, Figlin RA, Belldegrun AS. Renal cell carcinoma 2005: new frontiers in staging, prognostication and targeted molecular therapy. J Urol 2005;173:1853–62.[CrossRef][Medline]
  4. Cohen HT, McGovern FJ. Renal-cell carcinoma. N Engl J Med 2005;353:2477–90.[Free Full Text]
  5. Kovacs G, Akhtar M, Beckwith BJ, et al. The Heidelberg classification of renal cell tumours. J Pathol 1997;183:131–3.[CrossRef][Medline]
  6. Storkel S, Eble JN, Adlakha K, et al. Classification of renal cell carcinoma: Workgroup No. 1. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:987–9.[CrossRef][Medline]
  7. Latif F, Tory K, Gnarra J, et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 1993;260:1317–20.[Abstract/Free Full Text]
  8. Kondo K, Yao M, Yoshida M, et al. Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters. Genes Chromosomes Cancer 2002;34:58–68.[CrossRef][Medline]
  9. Linehan WM, Zbar B. Focus on kidney cancer. Cancer Cell 2004;6:223–8.[CrossRef][Medline]
  10. Kaelin WG, Jr. The von Hippel-Lindau tumor suppressor gene and kidney cancer. Clin Cancer Res 2004;10:6290–5S.
  11. Yao M, Tabuchi H, Nagashima Y, et al. Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma. J Pathol 2005;205:377–87.[CrossRef][Medline]
  12. Jiang HP, Serrero G. Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. Proc Natl Acad Sci U S A 1992;89:7856–60.[Abstract/Free Full Text]
  13. Londos C, Brasaemle DL, Schultz CJ, Segrest JP, Kimmel AR. Perilipins, ADRP, and other proteins that associate with intracellular neutral lipid droplets in animal cells. Semin Cell Dev Biol 1999;10:51–8.[CrossRef][Medline]
  14. Brasaemle DL, Barber T, Wolins NE, Serrero G, Blanchette-Mackie EJ, Londos C. Adipose differentiation-related protein is an ubiquitously expressed lipid storage droplet-associated protein. J Lipid Res 1997;38:2249–63.[Abstract]
  15. Gao J, Serrero G. Adipose differentiation related protein (ADRP) expressed in transfected COS-7 cells selectively stimulates long chain fatty acid uptake. J Biol Chem 1999;274:16825–30.[Abstract/Free Full Text]
  16. Frolov A, Petrescu A, Atshaves BP, et al. High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach. J Biol Chem 2000;275:12769–80.[Abstract/Free Full Text]
  17. Heid HW, Moll R, Schwetlick I, Rackwitz HR, Keenan TW. Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 1998;294:309–21.[CrossRef][Medline]
  18. Saarikoski ST, Rivera SP, Hankinsona O. Mitogen-inducible gene 6 (MIG-6), adipophilin and tuftelin are inducible by hypoxia. FEBS Lett 2002;530:186–90.[CrossRef][Medline]
  19. Childs R, Chernoff A, Contentin N, et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 2000;343:750–8.[Abstract/Free Full Text]
  20. Stadler WM. Targeted agents for the treatment of advanced renal cell carcinoma. Cancer 2005;104:2323–33.[CrossRef][Medline]
  21. Guinan P, Sobin LH, Algaba F, et al. TNM staging of renal cell carcinoma: Workgroup No. 3. Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer 1997;80:992–3.[CrossRef][Medline]
  22. Thoenes W, Storkel S, Rumpelt HJ. Histopathology and classification of renal cell tumors (adenomas, oncocytomas and carcinomas). The basic cytological and histopathological elements and their use for diagnostics. Pathol Res Pract 1986;181:125–43.[Medline]
  23. Shuin T, Kondo K, Torigoe S, et al. Frequent somatic mutations and loss of heterozygosity of the von Hippel-Lindau tumor suppressor gene in primary human renal cell carcinomas. Cancer Res 1994;54:2852–5.[Abstract/Free Full Text]
  24. Simon R. Roadmap for developing and validating therapeutically relevant genomic classifiers. J Clin Oncol 2005;23:7332–41.[Abstract/Free Full Text]
  25. Lossos IS, Czerwinski DK, Alizadeh AA, et al. Prediction of survival in diffuse large-B-cell lymphoma based on the expression of six genes. N Engl J Med 2004;350:1828–37.[Abstract/Free Full Text]
  26. Li G, Cuilleron M, Gentil-Perret A, et al. Rapid and sensitive detection of messenger RNA expression for molecular differential diagnosis of renal cell carcinoma. Clin Cancer Res 2003;9:6441–6.[Abstract/Free Full Text]
  27. Rosenwald A, Wright G, Chan WC, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med 2002;346:1937–47.[Abstract/Free Full Text]
  28. Cheville JC, Lohse CM, Zincke H, et al. Sarcomatoid renal cell carcinoma: an examination of underlying histologic subtype and an analysis of associations with patient outcome. Am J Surg Pathol 2004;28:435–41.[CrossRef][Medline]
  29. Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993;9:138–41.[CrossRef][Medline]
  30. Yokota J. Tumor progression and metastasis. Carcinogenesis 2000;21:497–503.[Abstract/Free Full Text]
  31. Baba M, Hirai S, Yamada-Okabe H, et al. Loss of von Hippel-Lindau protein causes cell density dependent deregulation of cyclinD1 expression through hypoxia-inducible factor. Oncogene 2003;22:2728–38.[CrossRef][Medline]
  32. Wiesener MS, Munchenhagen PM, Berger I, et al. Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1{alpha} in clear cell renal carcinomas. Cancer Res 2001;61:5215–22.[Abstract/Free Full Text]
  33. Leppert JT, Lam JS, Pantuck AJ, Figlin RA, Belldegrun AS. Carbonic anhydrase IX and the future of molecular markers in renal cell carcinoma. BJU Int 2005;96:281–5.[CrossRef][Medline]
  34. Togashi A, Katagiri T, Ashida S, et al. Hypoxia-inducible protein 2 (HIG2), a novel diagnostic marker for renal cell carcinoma and potential target for molecular therapy. Cancer Res 2005;65:4817–26.[Abstract/Free Full Text]
  35. Grabmaier K, A de Weijert MCA, Verhaegh GW, Schalken JA, Oosterwijk E. Strict regulation of CAIX (G250/MN) by HIF-1{alpha} in clear cell renal cell carcinoma. Oncogene 2004;23:5624–31.[CrossRef][Medline]
  36. Lamers CH, Sleijfer S, Vulto AG, et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol 2006;24:e20–2.[Free Full Text]
  37. Yao M, Yoshida M, Kishida T, et al. VHL tumor suppressor gene alterations associated with good prognosis in sporadic clear-cell renal carcinoma. J Natl Cancer Inst 2002;94:1569–75.[Abstract/Free Full Text]
  38. Targett-Adams P, McElwee MJ, Ehrenborg E, Gustafsson MC, Palmer CN, McLauchlan J. A PPAR response element regulates transcription of the gene for human adipose differentiation-related protein. Biochim Biophys Acta 2005;1728:95–104.[Medline]
  39. Desvergne B, Michalik L, Wahli W. Transcriptional regulation of metabolism. Physiol Rev 2006;86:465–514.[Abstract/Free Full Text]
  40. Mueller E, Sarraf P, Tontonoz P, et al. Terminal differentiation of human breast cancer through PPAR{gamma}. Mol Cell 1998;1:465–70.[CrossRef][Medline]
  41. Sarraf P, Mueller E, Jones D, et al. Differentiation and reversal of malignant changes in colon cancer through PPAR{gamma}. Nat Med 1998;4:1046–52.[CrossRef][Medline]
  42. Chang TH, Szabo E. Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor {gamma} in non-small cell lung cancer. Cancer Res 2000;60:1129–38.[Abstract/Free Full Text]
  43. Inoue K, Kawahito Y, Tsubouchi Y, et al. Expression of peroxisome proliferator-activated receptor {gamma} in renal cell carcinoma and growth inhibition by its agonists. Biochem Biophys Res Commun 2001;287:727–32.[CrossRef][Medline]
  44. Yuan J, Takahashi A, Masumori N, Itoh N, Tsukamoto T. Ligands for peroxisome proliferator-activated receptor {gamma} have potent antitumor effect against human renal cell carcinoma. Urology 2005;65:594–9.[CrossRef][Medline]
  45. Fidler IJ. The pathogenesis of cancer metastasis: the "seed and soil" hypothesis revisited. Nat Rev Cancer 2003;3:453–8.[CrossRef][Medline]
  46. Ramaswamy S, Ross KN, Lander ES, Golub TR. A molecular signature of metastasis in primary solid tumors. Nat Genet 2003;33:49–54.[CrossRef][Medline]
  47. Minn AJ, Gupta GP, Siegel PM, et al. Genes that mediate breast cancer metastasis to lung. Nature 2005;436:518–24.[CrossRef][Medline]



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