Purpose: Vascular cell adhesion molecule 1 (VCAM1) is a cell surface glycoprotein implicated in various pathophysiologic conditions. We measured VCAM1 expression levels in tumor tissues and evaluated its significance and prognostic use in renal cell carcinoma (RCC).

Experimental Design: We used real-time quantitative PCR to examine the VCAM1 expression levels of a total of 485 sporadic renal tumors, including 429 clear cell, 21 papillary, 17 chromophobe, 11 oncocytomas, and 7 collecting duct carcinomas. We retrospectively examined the relationship of this expression to various clinicopathologic variables and the von Hippel-Lindau alteration status. We evaluated its significance with respect to patient survival rates using the Cox regression model combined with the split-sample method.

Results: Compared with normal kidney samples (n = 43), VCAM1 was significantly up-regulated in clear cell RCC and papillary RCC, whereas it was down-regulated in chromophobe RCC and oncocytoma. In clear cell RCC, VCAM1 expression levels were apparently high in patients asymptomatic at presentation and in patients with small tumor size, low-stage, low-grade, microvascular invasion–negative, and von Hippel-Lindau alteration-positive tumors. Univariate analyses showed that VCAM1 high expression is strongly associated with better outcomes in clear cell and papillary RCCs. Further, Cox multivariate analysis models combined with the split-sample method revealed that this association is significant only in cancer-free survival for patients with clear cell RCC after curative surgical resection.

Conclusions:VCAM1 expression levels were found to be histologically subtype specific in renal tumors. Determination of the VCAM1 expression level as a biomarker can provide useful prognostic information for patients with clear cell RCC.

Renal cell carcinoma (RCC) is the most common malignant tumor of the adult kidney, accounting for ∼3% of human malignancies (1, 2). Although radiological imaging has revolutionized the diagnosis of RCC, nearly one third of patients present with metastatic disease at the time of diagnosis (1, 2). Additionally, as many as 40% of patients with local tumors will ultimately relapse with metastatic disease after curative surgical resection (2). New diagnostic and monitoring methods together with novel therapeutic agents are therefore essential for improving the outcome of patients with RCC (2, 3).

Recent studies have clearly shown that RCC is morphologically and genetically a mixture of heterogeneous tumors and can be classified as including at least four subtypes of tumors (411). An understanding of the detailed biology of each tumor subtype is therefore crucial for improving and developing “tailored medicine” for patients with RCC (7).

Microarray is a powerful tool that permits simultaneous rapid screening of expression levels for a large set of genes. With respect to microarray studies for RCC, several groups have reported the identification of sets of genes that are exclusively expressed in renal tumor subtypes or that may predict clinical outcomes based solely on gene expression levels (1217). Among these works, Vasselli et al. (13) have recently found that expression levels of vascular cell adhesion molecule 1 (VCAM1) show the highest statistically predictive value for the survival of patients with stage IV metastatic RCC. VCAM1 was originally identified as a cell adhesion molecule and is intimately involved in inflammatory reactions (18, 19). It is also involved in tumor progression, angiogenesis, and the metastatic process of malignant cells (20, 21). VCAM1 may play a role in tumor cell adhesion to the vascular endothelium, which precedes extravasation of cells and the development of metastases (20, 21). The up-regulation of VCAM1 and its prognostic significance have been reported in several human malignancies, including gastric, breast, and esophageal carcinomas (2225).

In the present study, to further elucidate the significance of VCAM1 as a biomolecular marker for RCC, we measured VCAM1 expression levels by real-time quantitative PCR and examined their relationship to various clinicopathologic variables as well as to patient outcomes in a large cohort of patients who had been followed up for a considerable length of time.

Renal tumors and patients. A total of 485 primary renal tumors, including 429 clear cell, 21 papillary, 17 chromophobe, and 7 collecting duct carcinomas plus 11 oncocytomas, was collected along with 43 normal kidney tissue samples from nonselected patients who consecutively underwent nephrectomy at Yokohama City University Hospital (Yokohama, Japan) and its affiliated hospitals between March 1986 and June 2004. Fresh tumor materials without apparent necrosis and normal kidney tissue specimens were grossly cut out, snap frozen with liquid nitrogen, and stored at −80°C until nucleic acid extraction. All patients were confirmed to have a sporadic disease based on their medical records. The histopathology of the tumors was classified according to standard classifications (4, 5). Tumor stage and grade were determined after surgical treatment according to the tumor-node-metastasis classification (26). We included 429 clear cell and 21 papillary RCC cases in the survival analysis. Of the 429 patients with clear cell RCC, presumably curative surgery was done in 337 patients, including 327 stage I to III patients (Unio Internationale Contra Cancrum classification) who underwent nephrectomy (311 conventional and 16 partial nephrectomies) and 10 stage IV patients with solitary metastasis who underwent nephrectomy together with curative metastasis resection. Cancer-free survival was therefore tested in this cohort of 337 patients. The remaining 92 patients with stage IV advanced disease received a palliative or adjunctive nephrectomy. Follow-up ended in October 2005, at which point the median follow-up periods after surgical treatment for clear cell RCC (n = 429) and papillary RCC cases (n = 21) were 68.6 and 58.4 months, respectively (Table 1). The study protocol was approved by the institutional review board of the Yokohama City University School of Medicine.

Table 1.

Characteristics of patients with clear cell RCC in the all-patient group, training set, and test set

CharacteristicAll patientsTraining setTest setP*
No. cases 429 181 248  
Nephrectomy period (y/mo) 86/3-04/6 86/3-94/10 94/11-04/6  
Age at nephrectomy (y)     
    Range 33-91 33-91 33-84 0.780 
    Mean 60.85 61.33 60.50  
    SD 10.77 10.62 10.89  
Sexual distribution, (%)     
    Female 119 (28) 50 (28) 69 (28) 1.000 
    Male 310 (72) 131 (72) 179 (72)  
Follow-up period for all cases (mo)     
    Range 0.4-235.6 1.0-235.6 0.4-129.8  
    Median 68.6 87.2 60.9  
Follow-up period for survivors (mo)     
    Number 303 117 186  
    Range 0.6-235.6 4.9-235.6 0.6-129.8  
    Median 83.6 118.8 74.4  
Symptom presentation, (%)     
    Negative 243 (57) 96 (53) 147 (59) 0.198 
    Positive 186 (43) 85 (47) 101 (41)  
Tumor size (cm)     
    Range 1.5-23 1.7-20 1.5-23 0.010§ 
    Mean 5.97 6.29 5.73  
    SD 3.43 3.28 3.52  
UICC stage, (%)     
    I 226 (53) 80 (44) 146 (59) 0.003§ 
    II 18 (4.2) 8 (4.4) 10 (4.0)  
    III 83 (19) 41 (23) 42 (17)  
    IV 102 (24) 52 (29) 50 (20)  
Tumor grade, (%)     
    G1 116 (27) 43 (24) 73 (29) 0.534§ 
    G2 185 (43) 85 (47) 100 (41)  
    G3 + G4 128 (30) 53 (29) 75 (30)  
Microvascular invasion, (%)     
    Negative 262 (61) 102 (56) 160 (65) 0.111 
    Positive 158 (37) 73 (40) 85 (34)  
    Not determined 9 (2.1) 6 (3.3) 3 (1.2)  
Recurrence after curative surgery (n = 337), (%)     
    No 267 (79) 105 (80) 162 (79) 0.845 
    Yes 70 (21) 26 (20) 44 (21)  
Cancer death, (%)     
    No 303 (71) 117 (65) 186 (75) 0.020 
    Yes 126 (29) 64 (35) 62 (25)  
VHL alteration (n = 219), (%)     
    Positive 116 (53) 87 (55) 29 (47) 0.315 
    Negative 103 (47) 70 (45) 33 (53)  
VCAM1 expression, (%)     
    Low 91 (21) 40 (22) 51 (21) 0.701 
    High 338 (79) 141 (78) 197 (79)  
CharacteristicAll patientsTraining setTest setP*
No. cases 429 181 248  
Nephrectomy period (y/mo) 86/3-04/6 86/3-94/10 94/11-04/6  
Age at nephrectomy (y)     
    Range 33-91 33-91 33-84 0.780 
    Mean 60.85 61.33 60.50  
    SD 10.77 10.62 10.89  
Sexual distribution, (%)     
    Female 119 (28) 50 (28) 69 (28) 1.000 
    Male 310 (72) 131 (72) 179 (72)  
Follow-up period for all cases (mo)     
    Range 0.4-235.6 1.0-235.6 0.4-129.8  
    Median 68.6 87.2 60.9  
Follow-up period for survivors (mo)     
    Number 303 117 186  
    Range 0.6-235.6 4.9-235.6 0.6-129.8  
    Median 83.6 118.8 74.4  
Symptom presentation, (%)     
    Negative 243 (57) 96 (53) 147 (59) 0.198 
    Positive 186 (43) 85 (47) 101 (41)  
Tumor size (cm)     
    Range 1.5-23 1.7-20 1.5-23 0.010§ 
    Mean 5.97 6.29 5.73  
    SD 3.43 3.28 3.52  
UICC stage, (%)     
    I 226 (53) 80 (44) 146 (59) 0.003§ 
    II 18 (4.2) 8 (4.4) 10 (4.0)  
    III 83 (19) 41 (23) 42 (17)  
    IV 102 (24) 52 (29) 50 (20)  
Tumor grade, (%)     
    G1 116 (27) 43 (24) 73 (29) 0.534§ 
    G2 185 (43) 85 (47) 100 (41)  
    G3 + G4 128 (30) 53 (29) 75 (30)  
Microvascular invasion, (%)     
    Negative 262 (61) 102 (56) 160 (65) 0.111 
    Positive 158 (37) 73 (40) 85 (34)  
    Not determined 9 (2.1) 6 (3.3) 3 (1.2)  
Recurrence after curative surgery (n = 337), (%)     
    No 267 (79) 105 (80) 162 (79) 0.845 
    Yes 70 (21) 26 (20) 44 (21)  
Cancer death, (%)     
    No 303 (71) 117 (65) 186 (75) 0.020 
    Yes 126 (29) 64 (35) 62 (25)  
VHL alteration (n = 219), (%)     
    Positive 116 (53) 87 (55) 29 (47) 0.315 
    Negative 103 (47) 70 (45) 33 (53)  
VCAM1 expression, (%)     
    Low 91 (21) 40 (22) 51 (21) 0.701 
    High 338 (79) 141 (78) 197 (79)  

Abbreviation: UICC, Unio Internationale Contra Cancrum.

*

Statistical test between training set and test set.

Independent t test.

χ2 test.

§

Mann-Whitney U test.

Measurement of VCAM1 expression by real-time quantitative PCR. Isolation of total RNA, cDNA preparation, and real-time quantitative PCR (RQ-PCR) with Taqman fluorescent probes for the measurement of VCAM1 expression were done essentially as described previously (27). The following primers and a probe for VCAM1 were used: 5′-CAAAGGCAGAGTACGCAAACAC-3′ (forward primer), 5′-CTGGCTCAAGCATGTCATATTCAC-3′ (reverse primer), 5′-6FAM-CAGAGATACAACCGTCTTGGTCAGCCCTT-TAMRA-3′ (probe). PCR amplification was done using the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The amount of product was measured by interpolation from a standard curve. In each experiment, at least two independent RQ-PCRs were done to obtain the mean expression signal values. The obtained signal values were normalized by dividing by the mean expression signal of β-actin (27).

Statistical analysis. The independent t test, Mann-Whitney U test, Kruskal-Wallis H test, or a χ2 test was used to determine and examine differences between groups. The correlations of the expression levels were examined by the Spearman rank correlation coefficient. In terms of the definition of cutoff values for VCAM1 expression level and 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. Survival time was defined as the time from nephrectomy or, in cases with recurrence after curative surgery, the time from the discovery of tumor recurrence until the patient's death or the last time at which the patient was known to be alive. 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 Cox regression models. In multivariate analysis, the Cox proportional hazards model was used to examine the simultaneous effects of several variables on patient outcome. All data were consistent with the assumptions of Cox proportional modeling. All statistical tests were two sided and were considered to be statistically significant at P < 0.05.

Up-regulation of VCAM1 expression in clear cell RCC and papillary RCC. We used RQ-PCR to analyze VCAM1 expression levels in a total of 485 primary renal tumors and 43 normal kidney tissue samples. We initially evaluated the reproducibility of the RQ-PCR assay. Of the samples included in the present analysis, we had previously examined the gene expression profiles of 41 samples using Human Genome U95A GeneChip microarrays (Affymetrix, Santa Clara, CA; ref. 27). When comparing the VCAM1 signal levels detected by the GeneChip (two corresponding probe sets, 41433_at and 583_s_at, on the platform) and RQ-PCR, we found that the reproducibility between these two procedures was quite high (Spearman rank correlation coefficient, ρ = 0.895 and 0.877, respectively).

As shown in Fig. 1, each tumor and normal kidney sample showed various VCAM1 expression levels in the RQ-PCR assay. When compared with normal kidney tissue samples (n = 43), both clear cell RCC and papillary RCC show apparently high VCAM1 expression levels. On the other hand, chromophobe RCCs and oncocytomas without exception showed very weak VCAM1 expression (Fig. 1).

Fig. 1.

Expression levels of VCAM1 by RQ-PCR according to the histologic subtype of renal tumors and normal kidney samples. VCAM1 expression levels were statistically significantly high in clear cell RCC and papillary RCC and low in chromophobe RCC and oncocytomas compared with normal kidney samples. Cl, clear cell RCC; Pap, papillary RCC; Pho, chromophobe RCC; Onc, oncocytoma; Cod, collecting duct carcinoma; Nrm, normal kidney tissue. P, Mann-Whitney U test.

Fig. 1.

Expression levels of VCAM1 by RQ-PCR according to the histologic subtype of renal tumors and normal kidney samples. VCAM1 expression levels were statistically significantly high in clear cell RCC and papillary RCC and low in chromophobe RCC and oncocytomas compared with normal kidney samples. Cl, clear cell RCC; Pap, papillary RCC; Pho, chromophobe RCC; Onc, oncocytoma; Cod, collecting duct carcinoma; Nrm, normal kidney tissue. P, Mann-Whitney U test.

Close modal

VCAM1 expression, various clinicopathologic features, and von Hippel-Lindau mutational status in clear cell RCC. Clear cell RCC was the most common histologic subtype overall; in addition, we found that VCAM1 is up-regulated in this subtype of tumor. We therefore focused primarily on clear cell RCC in the following study.

We first examined the correlation between VCAM1 expression levels and various clinicopathologic variables, finding the VCAM1 expression levels to be statistically significantly high in patients asymptomatic at presentation and in patients with smaller tumor size, lower stage, lower grading, or microvascular invasion-negative findings (Table 2).

Table 2.

Correlation between clinicopathologic characteristics and VHL alteration status and VCAM1 expression levels in patients with clear cell RCC

Characteristicn (%)VCAM1 expression level, median (interquartile range)P
Age (y)    
    ≤59 186 (43) 1.814 (2.429) 0.861* 
    ≥60 243 (57) 1.953 (2.501)  
Sexual distribution    
    Female 119 (28) 2.189 (2.370) 0.364* 
    Male 310 (72) 1.820 (2.460)  
Symptom presentation    
    Negative 243 (57) 2.163 (2.539) <0.001* 
    Positive 186 (43) 1.250 (2.614)  
Tumor size (cm)    
    ≤4.5 188 (44) 2.207 (2.456) 0.001 
    4.6-9.4 182 (43) 1.746 (2.228)  
    ≥9.5 59 (14) 1.060 (2.948)  
UICC stage    
    I 226 (53) 2.241 (2.433) <0.001 
    II 18 (4.2) 2.275 (2.066)  
    III 83 (19) 1.841 (2.753)  
    IV 102 (24) 0.988 (2.142)  
Tumor grade    
    G1 116 (27) 2.069 (2.070) <0.001 
    G2 185 (43) 2.113 (2.524)  
    G3 + G4 128 (30) 1.065 (2.871)  
Microvascular invasion    
    Negative + not determined 271 (63) 2.315 (2.330) <0.001* 
    Positive 158 (37) 1.472 (2.664)  
VHL alteration (n = 219)    
    Positive 116 (53) 2.131 (2.126) 0.001* 
    Negative 103 (47) 1.271 (2.017)  
Characteristicn (%)VCAM1 expression level, median (interquartile range)P
Age (y)    
    ≤59 186 (43) 1.814 (2.429) 0.861* 
    ≥60 243 (57) 1.953 (2.501)  
Sexual distribution    
    Female 119 (28) 2.189 (2.370) 0.364* 
    Male 310 (72) 1.820 (2.460)  
Symptom presentation    
    Negative 243 (57) 2.163 (2.539) <0.001* 
    Positive 186 (43) 1.250 (2.614)  
Tumor size (cm)    
    ≤4.5 188 (44) 2.207 (2.456) 0.001 
    4.6-9.4 182 (43) 1.746 (2.228)  
    ≥9.5 59 (14) 1.060 (2.948)  
UICC stage    
    I 226 (53) 2.241 (2.433) <0.001 
    II 18 (4.2) 2.275 (2.066)  
    III 83 (19) 1.841 (2.753)  
    IV 102 (24) 0.988 (2.142)  
Tumor grade    
    G1 116 (27) 2.069 (2.070) <0.001 
    G2 185 (43) 2.113 (2.524)  
    G3 + G4 128 (30) 1.065 (2.871)  
Microvascular invasion    
    Negative + not determined 271 (63) 2.315 (2.330) <0.001* 
    Positive 158 (37) 1.472 (2.664)  
VHL alteration (n = 219)    
    Positive 116 (53) 2.131 (2.126) 0.001* 
    Negative 103 (47) 1.271 (2.017)  
*

Mann-Whitney U test.

Kruskal-Wallis H test.

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is a critical genetic event for clear cell RCC. Among our 429 samples of clear cell RCCs, we had previously examined the VHL alteration status in 219 tumors and found that 116 (53%) showed a mutation of the VHL gene (28, 29). When we addressed VCAM1 expression and VHL gene status, we found that the expression levels were apparently high in VHL alteration-positive clear cell RCCs compared with VHL alteration-negative ones (P = 0.001, Mann-Whitney U test; Table 2).

VCAM1 expression and patient outcome in clear cell RCC. We next evaluated the effect of VCAM1 expression on survival rates for patients with clear cell RCC using the split-sample cross-validation approach. We divided the patients into two groups: the preliminary training set consisted of 181 patients treated in the earlier period (March 1986 to October 1994) and the validation test set consisted of the remaining 248 patients who were treated in the later period (November 1994 to June 2004; Table 1). We then determined the optimal cutoff value of the VCAM1 expression levels in the RQ-PCR assay in the training set group. As a result, the cutoff value was set at 0.770 and tumors whose VCAM1 expression was ≥0.770 were considered to be a high-expression tumor group, whereas the rest of the tumors were considered to be a low-expression group. Using the same method, tumor size was classified into three categories: ≤4.5, 4.6 to 9.4, and ≥9.5 cm (Supplementary Fig. S1). We then applied these cutoff values in the following survival tests.

In the present univariate analyses, the well-established prognostic variables, such as symptomatic presentation, tumor size, stage, grading, and microvascular invasion, were strongly associated with both cancer-specific survival and cancer-free survival in the training set, test set, and all patient cases. It is notable that VCAM1 expression levels were also statistically significantly correlated with survival in all of the patient cohorts examined and that patients with VCAM1 high-expressing clear cell RCC showed better outcomes than those with low-expression tumors (Fig. 2A and B; Supplementary Tables S1 and S2).

Fig. 2.

Kaplan-Meier survival probability and VCAM1 expression levels in the training set, test set, and all patients with clear cell RCC. The cancer-specific survival (A) and the cancer-free survival (B) of patients with clear cell RCC who underwent nephrectomy and the cancer-specific survival for advanced metastatic clear cell RCC cases (C). Numbers in parentheses, total numbers of cancer deaths (CD) and tumor recurrences (REC) in each group (A and B). For advanced metastatic RCC analyses, stage IV clear cell RCC patients who underwent noncurative surgery (n = 92) and patients in whom metastasis occurred after presumably curative nephrectomy (n = 70) were combined, and their survival probabilities were evaluated as a single cohort. P, log-rank test.

Fig. 2.

Kaplan-Meier survival probability and VCAM1 expression levels in the training set, test set, and all patients with clear cell RCC. The cancer-specific survival (A) and the cancer-free survival (B) of patients with clear cell RCC who underwent nephrectomy and the cancer-specific survival for advanced metastatic clear cell RCC cases (C). Numbers in parentheses, total numbers of cancer deaths (CD) and tumor recurrences (REC) in each group (A and B). For advanced metastatic RCC analyses, stage IV clear cell RCC patients who underwent noncurative surgery (n = 92) and patients in whom metastasis occurred after presumably curative nephrectomy (n = 70) were combined, and their survival probabilities were evaluated as a single cohort. P, log-rank test.

Close modal

To validate the prognostic effect of VCAM1, we next used multivariate analysis to determine whether the association between VCAM1 and patient survival was independent of other outcome predictors. The Cox models in the test set and all patient cases, however, failed to detect the independent prognostic value for cancer-specific survival tests (Table 3). On the other hand, for cancer-free survival, VCAM1 remained as an independent predictor both in the test set and in all patient cohorts (Table 4). These results strongly suggest that low expression of VCAM1 is a strong predictor of cancer recurrence for patients with clear cell RCC after curative surgical resection.

Table 3.

Cox multivariate analyses of cancer-specific survivals among patients with clear cell RCC in the test set and all patients

Test set (n = 248)
All patients (N = 429)
PHR (95% CI)PHR (95% CI)
Age (y)     
    ≤59  1.00  1.00 
    ≥60 0.416 1.27 (0.71-2.28) 0.219 1.27 (0.87-1.88) 
Sex     
    Female  1.00  1.00 
    Male 0.679 0.89 (0.52-1.53) 0.808 0.95 (0.64-1.42) 
Symptom     
    Negative    1.00 
    Positive 0.010 3.01 (1.29-7.00) 0.002 2.39 (1.37-4.19) 
Tumor size (cm)     
    ≤4.5  1.00  1.00 
    4.6-9.4 0.586 0.78 (0.31-1.93) 0.317 1.37 (0.74-2.54) 
    ≥9.5 0.661 0.80 (0.30-2.16) 0.109 1.74 (0.88-3.42) 
UICC stage     
    I + II  1.00  1.00 
    III 0.070 2.56 (0.93-7.09) 0.127 1.74 (0.85-3.53) 
    IV <0.001 6.92 (2.69-17.77) <0.001 7.18 (3.70-13.93) 
Tumor grade     
    G1  1.00  1.00 
    G2 0.098 3.54 (0.79-15.77) 0.049 2.27 (1.01-5.12) 
    G3 + G4 0.016 6.18 (1.40-27.36) 0.003 3.51 (1.55-7.93) 
Vascular invasion     
    Negative + not determined  1.00  1.00 
    Positive 0.039 2.06 (1.04-4.09) 0.011 1.80 (1.14-2.83) 
VCAM1     
    High  1.00  1.00 
    Low 0.789 0.92 (0.52-1.65) 0.190 1.29 (0.88-1.88) 
Test set (n = 248)
All patients (N = 429)
PHR (95% CI)PHR (95% CI)
Age (y)     
    ≤59  1.00  1.00 
    ≥60 0.416 1.27 (0.71-2.28) 0.219 1.27 (0.87-1.88) 
Sex     
    Female  1.00  1.00 
    Male 0.679 0.89 (0.52-1.53) 0.808 0.95 (0.64-1.42) 
Symptom     
    Negative    1.00 
    Positive 0.010 3.01 (1.29-7.00) 0.002 2.39 (1.37-4.19) 
Tumor size (cm)     
    ≤4.5  1.00  1.00 
    4.6-9.4 0.586 0.78 (0.31-1.93) 0.317 1.37 (0.74-2.54) 
    ≥9.5 0.661 0.80 (0.30-2.16) 0.109 1.74 (0.88-3.42) 
UICC stage     
    I + II  1.00  1.00 
    III 0.070 2.56 (0.93-7.09) 0.127 1.74 (0.85-3.53) 
    IV <0.001 6.92 (2.69-17.77) <0.001 7.18 (3.70-13.93) 
Tumor grade     
    G1  1.00  1.00 
    G2 0.098 3.54 (0.79-15.77) 0.049 2.27 (1.01-5.12) 
    G3 + G4 0.016 6.18 (1.40-27.36) 0.003 3.51 (1.55-7.93) 
Vascular invasion     
    Negative + not determined  1.00  1.00 
    Positive 0.039 2.06 (1.04-4.09) 0.011 1.80 (1.14-2.83) 
VCAM1     
    High  1.00  1.00 
    Low 0.789 0.92 (0.52-1.65) 0.190 1.29 (0.88-1.88) 

Abbreviations: HR, hazard ratio; 95% CI, 95% confidence interval.

Table 4.

Cox multivariate analyses of cancer-free survivals among patients with clear cell RCC treated with curative nephrectomy in the test set and all patients

CharacteristicTest set (n = 204)
All patients (N = 335)
PHR (95% CI)PHR (95% CI)
Age (y)     
    ≤59  1.00  1.00 
    ≥60 0.195 0.66 (0.35-1.24) 0.243 0.73 (0.43-1.24) 
Sex     
    Female  1.00  1.00 
    Male 0.557 1.24 (0.60-2.56) 0.055 1.72 (0.99-3.01) 
Symptom     
    Negative    1.00 
    Positive 0.633 1.19 (0.58-2.48) 0.092 1.59 (0.93-2.72) 
Tumor size (cm)     
    ≤4.5  1.00  1.00 
    4.6-9.4 0.064 2.34 (0.95-5.76) 0.007 2.76 (1.32-5.76) 
    ≥9.5 0.004 5.58 (1.72-18.07) <0.001 8.99 (3.49-23.12) 
UICC stage     
    I + II  1.00  1.00 
    III + IV 0.833 1.03 (0.77-1.37) 0.997 1.00 (0.81-1.23) 
Tumor grade     
    G1  1.00  1.00 
    G2 0.082 2.74 (0.88-8.55) 0.005 3.47 (1.44-8.35) 
    G3 + G4 0.002 5.98 (1.89-18.87) <0.001 5.26 (2.10-13.19) 
Vascular invasion     
    Negative + not determined  1.00  1.00 
    Positive 0.037 2.31 (1.05-5.10) 0.020 1.96 (1.11-3.44) 
VCAM1     
    High  1.00  1.00 
    Low 0.009 2.56 (1.27-5.16) 0.001 2.55 (1.44-4.49) 
CharacteristicTest set (n = 204)
All patients (N = 335)
PHR (95% CI)PHR (95% CI)
Age (y)     
    ≤59  1.00  1.00 
    ≥60 0.195 0.66 (0.35-1.24) 0.243 0.73 (0.43-1.24) 
Sex     
    Female  1.00  1.00 
    Male 0.557 1.24 (0.60-2.56) 0.055 1.72 (0.99-3.01) 
Symptom     
    Negative    1.00 
    Positive 0.633 1.19 (0.58-2.48) 0.092 1.59 (0.93-2.72) 
Tumor size (cm)     
    ≤4.5  1.00  1.00 
    4.6-9.4 0.064 2.34 (0.95-5.76) 0.007 2.76 (1.32-5.76) 
    ≥9.5 0.004 5.58 (1.72-18.07) <0.001 8.99 (3.49-23.12) 
UICC stage     
    I + II  1.00  1.00 
    III + IV 0.833 1.03 (0.77-1.37) 0.997 1.00 (0.81-1.23) 
Tumor grade     
    G1  1.00  1.00 
    G2 0.082 2.74 (0.88-8.55) 0.005 3.47 (1.44-8.35) 
    G3 + G4 0.002 5.98 (1.89-18.87) <0.001 5.26 (2.10-13.19) 
Vascular invasion     
    Negative + not determined  1.00  1.00 
    Positive 0.037 2.31 (1.05-5.10) 0.020 1.96 (1.11-3.44) 
VCAM1     
    High  1.00  1.00 
    Low 0.009 2.56 (1.27-5.16) 0.001 2.55 (1.44-4.49) 

VCAM1 survival analyses for advanced metastatic clear cell RCC. We further examined VCAM1 and cancer-specific survival for patients with advanced metastatic clear cell RCC because the prognostic effect of VCAM1 was originally reported in this patient cohort (13). In addition, the accurate prediction of survival time for patients with apparent tumor burdens is important for stratifying further treatment options (30). Our patient cohort included 92 stage IV patients who underwent palliative nephrectomy and 70 patients in whom tumor recurrence occurred after presumably curative nephrectomy. When they were combined and tested for survival, we detected a significant difference between the VCAM1 high- and low-expression groups in the training set and all cases but not in the test set group in the univariate analyses (Fig. 2C). In a further multivariate analysis model examining four variables (patient age, sex, tumor grading, and VCAM1 expression), VCAM1 was found to be an independent predictor in the training set but not in the test set or all cases (Supplementary Table S3).

VCAM1 survival analysis for papillary RCC. Finally, we evaluated the relationship between VCAM1 expression and survival effect in 21 patients with papillary RCC because papillary RCC also shows considerable VCAM1 expression levels. We adopted the same VCAM1 cutoff value (0.770) as in clear cell RCC cases. Kaplan-Meier analysis showed that high VCAM1 expression was associated with better cancer-specific survival and that the same cutoff value was applicable for this subtype of tumor (P = 0.011, log-rank test; Supplementary Fig. S2). Further multivariate analyses were not done due to the limited number of papillary RCC cases in the present cohort.

DNA microarray has been widely used to identify global gene expression signatures in various malignancies. In RCC, several studies have reported on the identification of tumor subtype-specific genes (14, 15, 17) or sets of genes corresponding to a predictable patient outcome (12, 13, 1517). However, several issues, such as technical complexity, the availability of high-quality RNA, and the cost of experiments at present, prevent its use as a routine expression detection technique. We therefore need simple and easy alternative methods of evaluating gene expression as well as ways of translating the knowledge obtained through microarray analysis to a routine clinical setting (31, 32). Our present strategy was to construct cDNA pools consisting of ∼500 renal tumors with various histologic subtypes together with corresponding normal kidney samples; we subsequently applied RQ-PCR to measure expression levels for important genes that would be applicable for novel diagnosis or therapeutic targets. As an initial step, we tried to validate the VCAM1 gene as a candidate prognostic marker in our RCC cDNA/RQ-PCR system. We found that our RQ-PCR system was not only simple and rapid but was also capable of preserving high reproducibility. Because our analysis was largely retrospective, we paid some attention to the study design. We first applied the split-sample method, a training test set approach, to confirm VCAM1 reproducibility and its predictive validity for patient survival rates (3234). It is known that RCCs tend to grow relatively slowly and that metastases sometimes occur >5 years after curative surgery unlike most other malignancies. Therefore, as the second focus of the present study, we assigned the patients treated in the early period to the training set group. This allowed us to assess the association of VCAM1 expression with patient clinical course in this group for a period of ≥11 years. As supposed, the cutoff value for VCAM1 detected by RQ-PCR was found to depend on the patient cohort. When we sought exact independent VCAM1 cutoff values for the training set, the test set, and the set of all patients, the detected cutoff values were found to differ slightly (0.770, 0.940, and 0.835, respectively). It is notable that the VCAM1 cutoff values detected in our model were very close to the expression levels for normal kidney tissue samples (median, 0.865; n = 43). The VCAM1 signal level in normal kidney tissue may be applicable as a reference cutoff value in an alternative model.

The prognostic significance of VCAM1 expression for RCC was originally reported by Vasselli et al. (13). They examined advanced metastatic RCCs using cDNA microarrays and found that the expression signature of a selected 45 probe sets can be predictable for the two patient groups (i.e., for the better and worse outcome groups). Moreover, the expression level of VCAM1 showed the highest statistically predictive value for survival among 6,400 gene probes on the array platforms. This incipient study, which was conducted for RCC patients with a restricted tumor stage (stage IV metastatic cases only) and which had a relatively small patient cohort (n = 58), prompted us to investigate the prognostic significance of VCAM1 in an independent patient cohort, including all tumor stages and a large number of patients with substantial follow-up periods. We determined that VCAM1 expression levels are specific to the histologic subtype of the tumor; both clear cell and papillary RCCs were found to express relatively high levels of VCAM1, whereas this expression was very weak in chromophobe RCC and oncocytomas. We found that clear cell and papillary RCC patients with tumors showing high VCAM1 expression had better survival rates than those with tumors showing low VCAM1 expression. It is particularly interesting that multivariate analyses combined with split-sample methods showed that this association was more statistically significant in cancer-free survival tests. Our data thus suggest that the determination of VCAM1 expression levels as a biomolecular marker can provide useful prognostic information for patients with clear cell RCC with respect to the prediction of cancer recurrence after curative surgical resection. On the other hand, unlike Vasselli et al. (13), we found that the VCAM1 prognostic value was fairly weak for advanced metastatic clear cell RCC cases. We detected its marginal significance only in the univariate analyses of subsets of the patient cohort. Various differences, including the mRNA detection method, subtraction cDNA microarray versus RQ-PCR, the definition of VCAM1 cutoff values, and the patient cohorts, might have been responsible for the discrepancy. Further validation in a larger patient cohort will be needed to clarify this point.

The up-regulation of VCAM1 was found to be associated with better outcome, including both low metastatic potential and slow tumor progression, in clear cell RCC. It is interesting that, in clear cell RCC, increased VCAM1 protein is observed primarily in cancer cells rather than in vascular endothelial cells by immunohistochemistry (13, 35, 36). In renal proximal tubular epithelial cells, which are considered to be a precursor to clear cell and papillary RCCs, VCAM1 remains at almost undetectable levels under normal conditions (13, 35, 36), although it has been induced in these cells under various pathologic conditions, such as acute renal allograft rejection, systemic lupus erythematosus, and glomerulonephritides (3739). VCAM1 binds to α4β1 and α4β7 integrins constitutively expressing on lymphocytes and macrophages and is responsible for adhering and recruiting these leukocytes to sites of active inflammation (40). It is notable that various degrees of leukocyte infiltration in cancerous regions are well characterized in RCC (41). Moreover, RCC is susceptible to immune-based treatments, such as IFNs, interleukin-2, and allogeneic stem cell transplantation (42, 43). VCAM1 shedding from RCC cells recruits these leukocytes to tumor tissues and may be involved in activation of the antitumor immune system, which leads to the suppression of tumor growth and metastasis.

We found that VCAM1 expression was apparently high in VHL alteration-positive clear cell RCCs compared with alteration-negative RCCs. VCAM1 transcription is regulated by the transcription factor nuclear factor-κB in renal proximal tubular epithelial cells (44), and activated nuclear factor-κB has been observed in many incidences of RCC (45, 46). An and Rettig (47) have recently shown that loss of VHL protein activates nuclear factor-κB via the hypoxia-inducible factor-α/transforming growth factor-α pathway, which subsequently activates an epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT/IκB kinase-α/nuclear factor-κB signaling cascade in RCC cells. Together, these data lead us to presume that one mechanism for the up-regulation of VCAM1 in clear cell RCC may be via the VHL/hypoxia-inducible factor/nuclear factor-κB pathway. In addition, we have previously shown that patients with VHL mutation–positive clear cell RCC show better survival compared with those with VHL mutation–negative clear cell RCC (48). The association between high VCAM1 expression and a good outcome may reflect, at least in part, the genetic features of the VHL alteration status in clear cell RCC.

In summary, we have constructed a cDNA pool/RQ-PCR system containing a large set of RCCs and found that the VCAM1 is a potent prognostic biomarker. Our preliminary findings undoubtedly should be further confirmed prospectively in a different patient cohort and using different detection methodologies, such as immunohistochemistry. We also plan to validate additional important genes that would be applicable to better diagnosis of RCC.

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

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Supplementary data