Hypoxia-inducible factor-1 (HIF-1), identified as one of the transcription factors, has been found to play an essential role in oxygen homeostasis. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β. Increased levels of HIF-1α have been reported during the carcinogenesis and progress of several tumors. We investigated the prognostic importance of HIF-1α expression in transitional cell carcinoma of the upper urinary tract. In 127 cases of transitional cell carcinoma of the upper urinary tract, we examined its expression (using immunohistochemistry and in situ hybridization), and also its relation to the expression of p53 oncoprotein, as well as to proliferating cell nuclear antigen (PCNA) immunoreactivity, microvessel density, clinicopathologic parameters, and clinical outcome. A positive expression of HIF-1α protein was recognized in 55.1% of samples, the expression being apparent within the nucleus in tumor cells. HIF-1α protein expression correlated with grade, growth pattern, p53 oncoprotein expression, and PCNA index, but not with stage. Furthermore, a significant correlation was found between HIF-1α protein expression and both overall and disease-free survival rates in the univariate and multivariate analyses (in all tumors and in invasive tumors). A positive expression of HIF-1α mRNA was recognized in 69.6% of 125 samples which were available, the expression being apparent within the cytoplasm in tumor cells. The positive expression of HIF-1α mRNA by in situ hybridization correlated significantly with HIF-1α protein expression by immunohistochemistry. HIF-1α mRNA expression only correlated with pattern of growth (P = 0.0078). In conclusion, the detection of HIF-1α protein would seem to be of value in informing the prognosis of transitional cell carcinoma of the upper urinary tract.

Angiogenesis, the growth of new blood vessels from those preexisting in the tissue, is essential for human tumor growth. It is now known that in the prevascular phase, with little or no angiogenic activity, solid tumors cannot expand beyond a few cubic millimeters. In contrast, the onset of angiogenic activity permits rapid expansion of the tumor, provided the level of such activity is sufficient to induce neovascularization. The metastasis of several types of carcinoma is associated with intense neovascularization, which may be a reflection of the angiogenic activity generated by tumor cells, and may thus be of use as a prognostic indicator (15).

A number of angiogenic factors has been identified over the last decade or so. Among these, some of the most important are vascular endothelial growth factor (VEGF), platelet-derived growth factor, and hypoxia-inducible factor-1 (HIF-1; refs. 68). Overexpressions of VEGF and platelet-derived growth factor are associated with angiogenesis and poor prognosis in several tumors, including transitional cell carcinoma (7, 913). HIF-1, identified as one of the transcription factors, has been found to play an essential role in oxygen homeostasis (14, 15). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β. HIF-1β, also known as aryl hydrocarbon receptor nuclear translocator, is constitutively expressed, and usually does not change following hypoxic stimulation. Under normoxic conditions, HIF-1α is maintained at low levels due to continuous degradation via the ubiquitin-dependent proteosome pathway, but this pathway is inhibited by hypoxia and by defects in the p53 or von Hippel-Lindau tumor-suppressor genes, leading to stabilization of the HIF-1α protein (1619). Thus, hypoxia leads to a rapid increase in HIF-1α protein levels (1416, 18). Furthermore, HIF-1α up-regulates a number of factors important for tumor expansion (20). Overexpression of HIF-1α protein has been found to be associated with tumor aggressiveness and with an unfavorable prognosis in several cancers, including transitional cell carcinoma of the urinary bladder (2134). In transitional cell carcinoma of the upper urinary tract, however, no study has yet been made of the relationship between HIF-1α protein expression and either tumor progression or prognosis.

In the present study, we examined the expressions of HIF-1α protein, p53 oncoprotein, and proliferating cell nuclear antigen (PCNA), as well as microvessel density in 127 cases of transitional cell carcinoma of the upper urinary tract. Our goal was to evaluate the diagnostic and prognostic importance of HIF-1α expression in transitional cell carcinoma of the upper urinary tract.

The material used comprised 127 surgically resected specimens from patients with primary transitional cell carcinoma of the upper urinary tract. These specimens had been obtained at the Mutual Aid Associations' Hospital, Tachikawa, and National Defense Medical College Hospital, Tokorozawa, between 1970 and 1995. Histopathologic stage was determined according to the criteria proposed by the International Union Against Cancer (34). Tumor cells were divided histopathologically into two grades using the criteria for urinary bladder tumors laid down by the Armed Forces Institute of Pathology (35).

For immunohistochemistry of HIF-1α protein, we used the polymer-peroxidase method (EnVision+/HRP, Dako Cytomation, Denmark) on deparaffinized sections, employing mouse monoclonal antibodies against HIF-1α protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:40. The sections received autoclave pretreatment with 0.05 mol/L citrate buffer (pH 6.0), for 20 minutes at 120°C before immunohistochemistry against the antibody. For the negative control, the incubation step with the primary antibody was omitted. Cells were considered immunoreactive when distinct nuclear staining was identified, as previously described (2931). The extent of staining was scored as: (−) indicating negative reaction of tumor cells; (±) <10% of tumor area stained; (+) 11% to 25% stained; (2+) 26% to 50% stained; or (3+) ≥51% stained. The tumors in which stained tumor cells made up >10% of the tumor were graded as positive. The evaluation was done twice by one investigator (K. Nakanishi) who was blind to both tumor stage and grade. Both p53 oncoprotein and PCNA were evaluated immunohistochemically; the technique used and the results obtained in these same patients have been reported elsewhere (36, 37). For the analysis of PCNA, the percentage of nuclei exhibiting a positive immunoreaction (PCNA index) was determined (on the basis of the immunoreaction in at least 1,000 tumor cells) as previously described (37). The PCNA index was classified as high if it was ≥69.5%, a figure representing the median value for the carcinomas. In addition, we measured microvessel density in 77 invasive carcinomas by immunohistochemistry, using a rabbit polyclonal antibody against factor VIII-related antigen (1:1,500, Dakopatts, Glostrup, Denmark). Microvessel density was assessed by light microscopy in those areas of tumor invasion containing the highest numbers of capillaries and small venules per unit area (neovascular “hotspots”), as previously described (3, 4). To this end, the areas of highest neovascularization were found by scanning the tumor sections at low magnification (×40) to identify those areas of tumor invasion having the greatest numbers of immunostained microvessels per unit area. After the areas of highest neovascularization had been identified, the number of microvessels was determined within individual ×200 fields (×20 objective and ×10 ocular; 0.739 mm2 per field). Any immunostained endothelial cell or endothelial cell cluster clearly separated from adjacent microvessels, tumor cells, and other connective tissue elements was considered to indicate a single, countable microvessel. Results were expressed for each tumor as the highest number of microvessels identified within any single ×200 field.

For Western blotting analysis, tissue samples were sonicated (UD200; Tomy Seiko, Tokyo, Japan) for 10 minutes in 500 μL of an ice-cold homogenizing buffer of the following composition: 8 mol/L urea, 10% glycerol, 10 mmol/L Tris-HCl (pH 6.8), 1% SDS, 5 mmol/L DTT, containing protease inhibitor cocktail (Complete Mini, Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany). An 800 g crude particulate fraction was discarded, and the supernatant was centrifuged at 15,000 × g for 60 minutes at 4°C. The protein concentration of each sample was determined by means of a BC protein assay system (Bio-Rad Laboratories, CA). Then, 200 μg amounts of protein extracts were separated by 7.5% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Hybond-N+, Amersham Pharmacia Biotech, Buckinghamshire, England). Membranes were blocked with 5% nonfat dry milk for 60 minutes at room temperature, then incubated overnight at 4°C with HIF antibody (Santa Cruz Biotechnology) diluted at 1:200. Finally, after blots had been incubated for 1 hour with horseradish peroxidase-conjugated secondary antibodies, they were visualized by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL).

For the reverse transcriptase-PCR (RT-PCR), total RNA was isolated using acid guanidinium isothiocyanate-phenol-chloroform extraction and ethanol precipitation. RT-PCR was done using an amplification reagent kit (TaqMan EZRT-PCR kit, Applied Biosystems, Alameda, CA) with HIF-1α or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. HIF-1α and GAPDH primers were synthesized by an automated DNA synthesizer. The sequences of the HIF-1α sense and antisense primers were 5′-CAA AAC ACA CAG CGA AGC TTT-3′ and 5′-CAT CCT GTA CTG TCC TGT GGT G-3′, respectively. The cDNA amplification product was predicted to be a 441-bp fragment in the cDNA of HIF-1α. Primers were also synthesized to amplify the cDNA encoding GAPDH, a constitutively expressed gene, as a control. For GAPDH, the sequences of the primers used were 5′-GAA GGT GAA GGT CGG AGT C-3′ (sense), 5′-GAA GAT GGT GAT GGG ATT TC-3′ (antisense). The reaction master mix was prepared according to the manufacturer's protocol to give final concentrations of 1× reaction buffer, 300 μmol/L dATP, 300 μmol/L dCTP, 300 μmol/L dGTP, 600 μmol/L dUTP, 3 mmol/L Mg(OAc)2, 0.1 units/μL rTth DNA polymerase, 0.01 units/μL AmpErase UNG, and 200 nmol/L primers. To perform PCR, the reverse transcriptase reaction was incubated at 60°C for 30 minutes, followed by incubation at 95°C for 5 minutes to deactivate AmpErase UNG. PCR was done using 40 amplification cycles at 95°C for 20 seconds and at 60°C for 1 minute using an ABI PRISM 7700 Sequence detector (Applied Biosystems). PCR products were separated by electrophoresis in a 3% agarose gel, and stained with ethidium bromide.

For in situ hybridization, deparaffinized 4% paraformaldehyde-fixed sections were treated with 0.2 N HCl for 20 minutes, then incubated in 2× SSC for 10 minutes at 37°C, and finally incubated in 5 μg/mL proteinase K for 10 minutes at 37°C. Sections were subsequently postfixed in 4% paraformaldehyde for 5 minutes, then incubated for 10 minutes in 0.1 mol/L triethanolamine buffer (pH 8.0), containing 0.25% (vol/vol) acetic anhydride to prevent nonspecific binding due to oxidation of the tissue. The HIF-1α cDNA probe used was a 441-bp fragment (the RT-PCR product mentioned above) subcloned into the EcoRI site of a pGEM-T Easy Vector (Promega, Madison, WI). Antisense probes and the corresponding sense probes were labeled with digoxigenin using SP6 and T7 polymerases, respectively, by means of an RNA labeling kit (Boehringer Mannheim, Mannheim, Germany). Hybridization was carried out overnight at 42°C in 50% (vol/vol) deionized formamide, 5× Denhardt's solution, 5% (wt/vol) dextran sulfate, 2× SSC, 0.3 mg/mL salmon sperm DNA, 5 mmol/L EDTA, and 0.01 μg/mL digoxigenin-labeled probes. After performing a final stringency wash at 37°C for 20 minutes, hybridization was detected immunologically. The extent of cytoplasmic staining was scored as: (−) indicating negative reaction of tumor cells; (±) <10% of tumor area stained; (+) 11% to 25% stained; (2+) 26% to 50% stained; or (3+) ≥51% stained. The tumors in which stained tumor cells made up >10% of the tumor were graded as positive.

For statistical analysis, disease-free and overall survival rates were the two main dependent variables tested in this study. “Disease-free survival” was defined as the period between the initial radical operation and the subsequent appearance of recurrence or metastasis. Recurrence was defined as TCC occurring anywhere in the genitourinary tract. The endpoint was either recurrence/metastasis of TCC or the closing date of the study, whichever came first. “Overall survival” was defined as the interval between surgery and death, the end-point for this variable was either death or the closing date of the study.

Disease-free and overall survival curves for all of the univariate analyses were assessed using the Kaplan-Meier method. Comparisons between two or more survival curves were assessed using Wilcoxon and log-rank test. Multivariate analysis of the clinicopathologic parameters was done using the Cox stepwise-regression model. The above analyses were done using the SAS statistical software package (SAS Institute, Inc., Carey, NC; ref. 38). The comparison, with respect to stage in all tumors, was done using the Kruskal-Wallis test. Comparisons with respect to age, sex, grade, pattern of growth, p53 oncoprotein, and PCNA index were done using the χ2 analysis.

The clinicopathologic findings for 127 patients with transitional cell carcinoma of the upper urinary tract are shown in Table 1. The patients' ages at diagnosis were within the range 34 to 84 years, with a median age of 67 years. Thirty-one of the 127 patients died as a result of their tumors 1 to 132 months after surgery (mean, 24 months; median, 15 months). The remainder survived 0 to 257 months after surgery (mean, 70 months; median 64 months).

Table 1.

Clinicopathologic findings in 127 patients with transitional cell carcinoma of the upper urinary tract

Survival (months)
Clinicopathologic findingsNo. of casesMeanMedianRange
Original site     
    Renal pelvic or calyces 53 61 37 0-257 
    Ureter 50 65 59 4-243 
    Multiple 24 41 28 1-120 
Associated bladder tumors 39    
    Simultaneous bladder tumors 11 33 13 1-110 
    Subsequent bladder tumors 29 57 37 7-175 
    Antecedent bladder tumors 38 44 5-66 
    Tumors at more than one of the above times 21 14 13-43 
Initial management* 120    
    Complete nephroureterectomy with a bladder cuff 90 58 36 1-203 
    Nephroureterectomy without a bladder cuff 71 47 4-257 
    Nephroureterectomy with total cystectomy 10 55 33 0-186 
    Nephrectomy 11 74 37 5-243 
    Urectomy 93   
Adjacent chemotherapy 31    
    Chemotherapy 21 59 64 2-161 
    Radiotherapy 38 19 7-117 
    Both 32 44 8-60 
Stage     
    Group A 48    
        Patients with recurrence, metastasis, or both 10 40 15 6-123 
        Patients without recurrence, metastasis, or both 38 84 87 4-203 
    Groups B and C 79    
        Patients with recurrence, metastasis, or both 37 37 15 1-175 
        Patients without recurrence, metastasis, or both 37 57 34 0-257 
        Patients who had metastasis at surgery or the tumor could not be excised totally by surgery 48 37 4-93 
Survival (months)
Clinicopathologic findingsNo. of casesMeanMedianRange
Original site     
    Renal pelvic or calyces 53 61 37 0-257 
    Ureter 50 65 59 4-243 
    Multiple 24 41 28 1-120 
Associated bladder tumors 39    
    Simultaneous bladder tumors 11 33 13 1-110 
    Subsequent bladder tumors 29 57 37 7-175 
    Antecedent bladder tumors 38 44 5-66 
    Tumors at more than one of the above times 21 14 13-43 
Initial management* 120    
    Complete nephroureterectomy with a bladder cuff 90 58 36 1-203 
    Nephroureterectomy without a bladder cuff 71 47 4-257 
    Nephroureterectomy with total cystectomy 10 55 33 0-186 
    Nephrectomy 11 74 37 5-243 
    Urectomy 93   
Adjacent chemotherapy 31    
    Chemotherapy 21 59 64 2-161 
    Radiotherapy 38 19 7-117 
    Both 32 44 8-60 
Stage     
    Group A 48    
        Patients with recurrence, metastasis, or both 10 40 15 6-123 
        Patients without recurrence, metastasis, or both 38 84 87 4-203 
    Groups B and C 79    
        Patients with recurrence, metastasis, or both 37 37 15 1-175 
        Patients without recurrence, metastasis, or both 37 57 34 0-257 
        Patients who had metastasis at surgery or the tumor could not be excised totally by surgery 48 37 4-93 
*

Patients were not treated as having bladder cancer.

The tumors were divided into three groups on the basis of tumor stage (A, papillary, noninvasive tumors, pTa; B, tumors invading the submucosa or muscularis, pT1 and pT2; and C, tumors invading beyond the muscularis or renal parenchyma, or metastasizing the regional lymph node or a distant site, pT3 and pT4).

When cells were classified as “immunoreactive” if distinct nuclear staining was identified, a positive expression of HIF-1α protein was recognized in 70 (55.1%) of the samples (Fig. 1A). A weak-to-moderate HIF-1α protein expression was also observed within the cytoplasm of tumor cells. In the normal urothelium, its expression was not detected. In our Western blotting analysis, HIF-1α protein was detected as one band in tumor tissue (Fig. 2A). A positive expression of p53 oncoprotein was recognized in 34 (26.7%) of the patients. The immunoreactivity for this oncoprotein was confined to the nuclei of tumor cells; no cytoplasmic staining was observed. A positive expression of PCNA was recognized in all tumors, the immunoreactivity being confined to tumor cell nuclei. The PCNA index was within the range 7.4% to 93.0% (mean and median values, 66.2% and 69.7%, respectively).

Fig. 1.

HIF-1α expression (A, protein; B, mRNA) in transitional cell carcinoma of the upper urinary tract. Bar, 100 μm.

Fig. 1.

HIF-1α expression (A, protein; B, mRNA) in transitional cell carcinoma of the upper urinary tract. Bar, 100 μm.

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Fig. 2.

Western blot analysis and RT-PCR results for HIF-1α in normal pelvic tissue and transitional cell carcinoma. A, HIF-1α protein was detected as one band, moderately in the tumor, but only weakly in normal pelvic tissue. B, PCR products consistent with the predicted PCR fragment for HIF-1α mRNA were detected in both tissues.

Fig. 2.

Western blot analysis and RT-PCR results for HIF-1α in normal pelvic tissue and transitional cell carcinoma. A, HIF-1α protein was detected as one band, moderately in the tumor, but only weakly in normal pelvic tissue. B, PCR products consistent with the predicted PCR fragment for HIF-1α mRNA were detected in both tissues.

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In our assessment of whether HIF-1α protein expression was related to p53 oncoprotein expression, PCNA index, or clinicopathologic findings, HIF-1α protein expression was found to be associated with grade, pattern of growth, p53 expression, and PCNA index, but not with stage (Table 2). When HIF-1α protein expression was assessed separately for noninvasive and invasive tumors, a significant correlation with HIF-1α protein expression was found for age (P = 0.034) and PCNA index (P = 0.013) in noninvasive tumors, for grade (P = 0.0077), and for p53 oncoprotein expression (P = 0.0038) in invasive tumors (Table 2). In invasive tumors, however, no significant relationship was found between HIF-1α protein expression and microvessel density (Table 2).

Table 2.

Relationship between HIF-1α immunoreactivity and other tumor characteristics (clinicopathologic findings, p53 oncoprotein immunoreactivity, PCNA index, and microvessel density) in 127 cases

All tumors (n = 127)
Noninvasive tumors (n = 48)
Invasive tumors (n = 79)
Correlative dataNo of casesImmunoreactivity of HIF-1αPNo of casesImmunoreactivity of HIF-1αPNo of casesImmunoreactivity of HIF-1αP
Age          
    <67 66 39 0.34 26 15 0.034 40 24 0.70 
    >67 61 31  22  39 25  
Sex          
    Male 93 48 0.18 38 17 0.78 55 31 0.11 
    Female 34 22  10  24 18  
Stage*          
    Group A 48 21 0.16 48 21     
    Group B 22 14     22 14 0.85 
    Group C 57 35     57 35  
Grade          
    Low 78 35 0.0034 43 19 0.85 35 16 0.0077 
    High 49 35   44 33  
Pattern of growth          
    Papillary 90 44 0.027    42 23 0.15 
    Nonpapillary 37 26     37 26  
p53 oncoprotein expression          
    Positive reaction 34 28 0.0002 0.084 29 24 0.0038 
    Negative reaction 93 42  43 17  50 25  
PCNA index          
    <69.5% 63 28 0.016 30 0.013 33 19 0.49 
    ≥69.5% 64 42  18 12  46 30  
Microvessel density          
    <70       39 23 0.58 
    ≥70       40 26  
All tumors (n = 127)
Noninvasive tumors (n = 48)
Invasive tumors (n = 79)
Correlative dataNo of casesImmunoreactivity of HIF-1αPNo of casesImmunoreactivity of HIF-1αPNo of casesImmunoreactivity of HIF-1αP
Age          
    <67 66 39 0.34 26 15 0.034 40 24 0.70 
    >67 61 31  22  39 25  
Sex          
    Male 93 48 0.18 38 17 0.78 55 31 0.11 
    Female 34 22  10  24 18  
Stage*          
    Group A 48 21 0.16 48 21     
    Group B 22 14     22 14 0.85 
    Group C 57 35     57 35  
Grade          
    Low 78 35 0.0034 43 19 0.85 35 16 0.0077 
    High 49 35   44 33  
Pattern of growth          
    Papillary 90 44 0.027    42 23 0.15 
    Nonpapillary 37 26     37 26  
p53 oncoprotein expression          
    Positive reaction 34 28 0.0002 0.084 29 24 0.0038 
    Negative reaction 93 42  43 17  50 25  
PCNA index          
    <69.5% 63 28 0.016 30 0.013 33 19 0.49 
    ≥69.5% 64 42  18 12  46 30  
Microvessel density          
    <70       39 23 0.58 
    ≥70       40 26  

NOTE: Comparisons (for all data except stage) were done using the χ2 analysis. The comparison with respect to stage in all tumors was done using the Kruskal-Wallis test.

*

The tumors were divided into three groups on the basis of tumor stage (A, papillary, noninvasive tumors, pTa; B, tumors invading the submucosa or muscularis, pT1 and pT2; and C, tumors invading beyond the muscularis or renal parenchyma, or metastasizing the regional lymph node or a distant site, pT3 and pT4).

By in situ hybridization, HIF-1α mRNA was detected within the cytoplasm of tumor cells (Fig. 1B). A positive expression of HIF-1α mRNA was recognized in 87 (69.6%) of the 125 samples that were available. In the normal urothelium, its expression was detected only weakly (data not shown). By RT-PCR, HIF-1α mRNA was detected on a 3% agarose gel in both tumor and normal pelvic tissues (Fig. 2B). The positive expression of HIF-1α mRNA by in situ hybridization correlated significantly with HIF-1α protein expression by immunohistochemistry (P < 0.0001). In our assessment of whether HIF-1α mRNA expression was related to p53 oncoprotein expression, PCNA index, or clinicopathologic findings, however, HIF-1α mRNA expression was only found to be associated with pattern of growth (P = 0.0078).

Assessment of distant metastasis–free interval and overall survival revealed rates for a 5-year disease-free survival and a 5-year overall survival of 61.1% and 72.1%, respectively. In the assessment of disease-free survival, 122 patients who had no metastasis at surgery and in whom the malignant tumor was excised totally by surgery were included in the analysis. In the assessment of overall survival, all 127 patients were included in the analysis. Univariate analyses of disease-free and overall survival rates in all patients revealed that HIF-1α protein expression, p53 oncoprotein expression, PCNA index, stage, grade, and pattern of growth, all had a significant effect on each of the two survival rates in all tumors (Table 3; Fig. 3). In all patients with HIF-1α protein-reactive tumors, the disease-free and overall survival rates were reduced by 9.7- and 5.0-fold, respectively (versus those for patients with HIF-1α protein-nonreactive tumors). In patients with invasive tumors, HIF-1α protein expression, p53 oncoprotein expression, and stage had a significant effect on disease-free survival rate, whereas HIF-1α protein expression and stage had a significant effect on overall survival rate. In patients with HIF-1α protein-reactive tumors, the disease-free and overall survival rates were reduced by 9.7- and 6.5-fold, respectively (versus those for patients with HIF-1α protein-nonreactive tumors). In the final models of the multivariate analysis for all tumors, stage and HIF-1α protein expression were shown to be prognostic factors for disease-free and overall survival rates: for disease-free survival, the values obtained for risk ratio were 7.78 (P < 0.0001) and 6.08 (P = 0.0009), respectively; for overall survival, they were 9.04 (P < 0.0001) and 4.87 (P = 0.0013), respectively. In invasive tumors, stage and HIF-1α protein expression were shown to be prognostic factors for disease-free and overall survival rates: for disease-free survival, the values obtained for risk ratio were 3.67 (P = 0.017) and 11.44 (P = 0.0010), respectively; for overall survival, they were 4.49 (P = 0.011) and 7.42 (P = 0.0011), respectively.

Table 3.

Univariate analysis of overall and disease-free survival rates

All tumors
Noninvasive tumors
Invasive tumors
Overall survival (n = 127)
Disease-free survival (n = 122)
Overall survival (n = 48)
Disease-free survival (n = 48)
Overall survival (n = 79)
Disease-free survival (n = 74)
Prognostic indicatorWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rank
HIF-1α 0.0001 0.0002 0.0001 0.0002 0.25 0.20 0.26 0.19 0.0004 0.0004 0.0003 0.0001 
p53 oncoprotein 0.018 0.0028 0.0014 0.0002 0.65 0.60 0.68 0.63 0.46 0.13 0.10 0.029 
PCNA index 0.011 0.0079 0.073 0.015 0.83 0.54 0.66 0.61 0.10 0.096 0.45 0.16 
Microvessel density 0.80 0.94 0.79 0.89 
Stage 0.0001 0.0001 0.0001 0.0001 0.013 0.015 0.089 0.045 
Grade 0.0002 0.0003 0.0004 0.0002 0.72 0.72 0.72 0.72 0.071 0.10 0.14 0.10 
Pattern of growth 0.0026 0.017 0.0044 0.012 0.44 0.95 0.53 0.90 
All tumors
Noninvasive tumors
Invasive tumors
Overall survival (n = 127)
Disease-free survival (n = 122)
Overall survival (n = 48)
Disease-free survival (n = 48)
Overall survival (n = 79)
Disease-free survival (n = 74)
Prognostic indicatorWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rankWilcoxonLog rank
HIF-1α 0.0001 0.0002 0.0001 0.0002 0.25 0.20 0.26 0.19 0.0004 0.0004 0.0003 0.0001 
p53 oncoprotein 0.018 0.0028 0.0014 0.0002 0.65 0.60 0.68 0.63 0.46 0.13 0.10 0.029 
PCNA index 0.011 0.0079 0.073 0.015 0.83 0.54 0.66 0.61 0.10 0.096 0.45 0.16 
Microvessel density 0.80 0.94 0.79 0.89 
Stage 0.0001 0.0001 0.0001 0.0001 0.013 0.015 0.089 0.045 
Grade 0.0002 0.0003 0.0004 0.0002 0.72 0.72 0.72 0.72 0.071 0.10 0.14 0.10 
Pattern of growth 0.0026 0.017 0.0044 0.012 0.44 0.95 0.53 0.90 
*

P value was not determined because of one factor or because that particular analysis was not done.

Fig. 3.

Disease-free and overall survival curves for all patients with transitional cell carcinoma (subdivided according to HIF-1α protein expression status). A, disease-free survival curve: patients with negative (n = 57) or positive (n = 70) HIF-1α protein expression. B, overall survival curve: patients with negative (n = 55) or positive (n = 67) HIF-1α protein expression.

Fig. 3.

Disease-free and overall survival curves for all patients with transitional cell carcinoma (subdivided according to HIF-1α protein expression status). A, disease-free survival curve: patients with negative (n = 57) or positive (n = 70) HIF-1α protein expression. B, overall survival curve: patients with negative (n = 55) or positive (n = 67) HIF-1α protein expression.

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The purpose of our investigation was to look for possible relations between HIF-1α protein and mRNA expressions and p53 oncoprotein expression, PCNA index, microvessel density, clinicopathologic findings, or clinical outcome in transitional cell carcinoma of the upper urinary tract. Our analysis revealed positive relationships between HIF-1α protein expression and p53 oncoprotein expression, PCNA index, grade, and pattern of growth, but no correlation between HIF-1α protein expression and stage. Furthermore, significant correlations were found between HIF-1α protein expression and both overall and disease-free survival rates in the univariate and multivariate analyses (in all tumors and in invasive tumors). Thus, the detection of the active form of HIF-1α protein would seem to be of value in informing the prognosis in transitional cell carcinoma of the upper urinary tract.

In urothelial carcinoma, differences in the anatomy of tumors result in differences in survival. Although the prognostic significance has been established for both stage and grade (39, 40), it is important to identify prognostic markers that will predict which patients are likely to have disease progression. Patients who have a subsequent intravesical recurrence can be treated with more effective adjuvant therapy. Several markers, including epithelial growth factor receptor, p53, and E-cadherin, are known to be associated with progression and prognosis in transitional cell carcinoma of the upper urinary tract (36, 4143). Recently, angiogenesis has also been shown to be associated with prognosis (9). With regard to HIF-1α protein, its high expression has been found to be associated with both tumor aggressiveness and an unfavorable prognosis in a variety of tumors, including carcinomas of the uterine cervix, colon, ovary, lung, oropharyngus, and breast (2133). In urinary tract carcinoma, we know of only two published reports on HIF-1α (34, 44). In one of those papers, Jones et al. (44)—who examined the expression of HIF-1α and HIF-2α mRNAs and proteins in 12 urinary bladder cancer and 4 normal bladder specimens using an RNase protection assay and immunoblot analysis—noted that expressions of HIF-1α and HIF-2α mRNAs and proteins were up-regulated in primary bladder tumors (by comparison with normal bladder specimens). With regard to the relationship between the expression of HIF-1α protein and clinicopathologic findings or outcome in urinary tract carcinoma, Theodoropoulos et al. (33)—who carried out an immunohistochemical examination in 93 patients with urothelial bladder cancer—showed that HIF-1α could be recognized through the nuclear staining of positive cells; in addition, they found a significant positive association with grade. Furthermore, patients characterized by HIF-1α overexpression had significantly worse overall and disease-free survival rates in their univariate and multivariate analyses. However, no published report has examined the relation between the expression of HIF-1α and clinicopathologic findings or survival rates in upper urinary tract tumors. In our immunohistochemical examination, expression of HIF-1α protein in tumor cells was detected within the nucleus and/or within the cytoplasm, which is in accordance with the staining patterns reported in several types of tumors (2133). Since it can be assumed that nuclear HIF-1α protein is the active form, our analysis was based only on the nuclear expression, as in previous reports (2931). In fact, our study of the expression of HIF-1α mRNA failed to find an association with either progression or prognosis. Although HIF-1α mRNA expression (by in situ hybridization) correlated significantly with its protein expression (by immunohistochemistry), its mRNA expression may not correlate with the active form of nuclear HIF-1α protein. In our study, expression of HIF-1α protein was found to be associated with grade, pattern of growth, p53 oncoprotein expression, and PCNA index in all tumors, although not with stage. Furthermore, its expression was associated with age and PCNA index in noninvasive tumors, and with grade and PCNA index in invasive tumors. We also found a significant correlation between HIF-1α protein expression and clinical outcome in the univariate and multivariate analyses of disease-free and overall survival rates, although no correlation between HIF-1α protein expression and stage was found. On this basis, the detection of HIF-1α protein would seem to be of value in predicting tumor prognosis in transitional cell carcinoma of the upper urinary tract.

It is known that certain features of tumor anatomy and of the biological behavior of tumor HIF-1α activate the transcription of the genes encoding VEGF, endothelin-1, and inducible nitric oxide synthase. In several tumors, strong associations of HIF-1α protein expression with angiogenic factors and higher microvessel densities have been reported (2225, 27, 44). In transitional cell carcinomas of the urinary bladder, Theodoropoulos et al. observed that HIF-1α protein correlated significantly with VEGF expression and microvessel density. In our study, however, we failed to find an association between HIF-1α protein and mRNA expressions and microvessel density. A similar lack of association has been noted in previous studies of oropharyngeal cancer and head and neck cancers (24, 25, 29). We are not entirely surprised at this lack of a direct correlation between HIF-1α protein and mRNA expressions and microvessel density because of the diversity of the other biological processes involved in the promotion of angiogenesis.

It is well known that wild-type p53 induces apoptosis (45). Furthermore, hypoxia may induce p53-dependent apoptosis and inhibit tumor growth (46, 47). Fairly recently, An et al. (19) reported that HIF-1α increases the stability of p53 protein and that a loss of wil49d-type p53 is associated with a marked reduction in hypoxia-mediated apoptosis. Both HIF-1α overexpression and p53 protein dysfunction would seem to be necessary for HIF-1α to exert a sufficient stimulation of tumor progression in early cancerogenesis (through angiogenesis and the induction of adaptive intracellular responses to hypoxia without a supporting proapoptotic mechanism). Zhong et al. (30) showed that HIF-1α overexpression is associated with aberrant p53 accumulation in 75 human cancers, including the majority of colon and breast cancers. Since the aberrant forms of p53 are inactive compared with the wild-type, they suggested that in addition to the link between HIF-1α and hypoxia, an inactivation of tumor-suppressor genes is associated with increased HIF-1α expression. In the present study of transitional cell carcinoma of the upper urinary tract, we also showed a significant correlation between the expression of HIF-1α protein and that of p53 oncoprotein. Since the half-life of the wild-type protein is too short for it to be detected by immunohistochemistry, any detection of p53 oncoprotein immunoreactivity is considered to be indirect evidence of p53 gene mutation (47). Our result is therefore consistent with the above suggestion of Zhong et al. (30).

In conclusion, the finding of a high expression of HIF-1α protein may provide information about the prognosis in cases of transitional cell carcinoma of the upper urinary tract. In the last few years, several investigators have shown HIF-2α expression in tumor-associated macrophages, and they have noted that the critical determinant of HIF activity is the respective levels of HIF-1α and HIF-2α proteins (16, 48, 49). Furthermore, an expression of HIF-2α protein is associated with a poor prognosis in non–small cell lung carcinoma and breast cancer (25, 50). On the other hand, it is well known that HIF-1α is an important mediator of the response of tumor cells to hypoxia, and that it up-regulates VEGF expression (8, 13, 17, 19, 2128). The VEGF gene contains a number of HIF1-binding sites in its regulatory region, and HIF-1α has been shown to activate the VEGF promoter in vitro (51, 52). Therefore, future studies should examine HIF-2α expression and VEGF expression, and seek to elucidate the relationship between their expressions and clinicopathologic findings or clinical outcome in transitional cell carcinoma of the upper urinary tract.

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.

We thank Dr. R. Timms for correcting the English.

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