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© 2000 American Society for Clinical Oncology Independent Prognostic Value of Serum Hepatocyte Growth Factor in Bladder CancerFrom the Department of Urology, Osaka Medical College, Takatsuki; Department of Urology, Hyogo Medical Center for Adults, Akashi; Otsuka Pharmaceutical Co, Ltd, Tokushima; Department of Pathology, Kobe University School of Medicine, Kobe; and Biology Research, Tsukuba Research Institute Novartis Pharmaceuticals K.K., Tsukuba, Japan. Address reprint requests to Kazuo Gohji, MD, PhD, Department of Urology, Osaka Medical College, 2–7 Daigakumatchi, Takatsuki, Osaka, 569-8686 Japan; email uro009{at}poh.osaka-med.ac.jp
PURPOSE: We retrospectively investigated whether the level of serum hepatocyte growth factor could predict the prognosis and extent of transitional-cell carcinoma of the urinary bladder. PATIENTS AND METHODS: Serum samples were collected from 113 patients with bladder cancer and from 200 healthy controls. Of the 113 patients, 59 had superficial bladder cancer and 54 had muscle-invasive cancer. Thirteen bladder cancer tissues (eight superficial and five muscle-invasive) were also collected. The levels of hepatocyte growth factor in the serum and tissues of these individuals were measured by enzyme-linked immunoadsorbent assay using hepatocyte growth factor antibodies. RESULTS: The levels of hepatocyte growth factor in the serum and tissues of patients with muscle-invasive cancer were significantly higher than those of patients with superficial bladder cancer (P < .0001 and P = .0054, respectively). The degree of elevation above the normal level of serum hepatocyte growth factor of the former (61.1%) was significantly higher than that of the latter (8.4%; P < .0001). The elevation was highest in patients with visceral metastasis (93.3%). Among patients with superficial bladder cancer, the overall survival rate of those with low levels of serum hepatocyte growth factor was significantly greater than that of those with high levels (P = .005). Among patients with minimally invasive bladder cancer, the disease-free and overall survival rates of those with high levels of serum hepatocyte growth factor were significantly lower than the same rates of those with low levels (P < .001 and P = .0028, respectively). CONCLUSION: Our study suggests that the level of hepatocyte growth factor in serum could be a predictor of patient survival and extent of bladder cancer.
BLADDER CANCER IS the fourth most common malignant tumor in men and the eighth most common cancer in women in the United States.1 Approximately 70% to 80% of bladder cancers are superficial, whereas the remainder are muscle-invasive or metastatic cancers. Several prognostic factors and predictors of disease extension in bladder cancer tissue and urine have been reported.2-4 Bladder cancer patients who exhibit high expression of autocrine motility factor receptors have significantly lower survival rates than patients with low expression.2 Superficial bladder cancers with high expression of vascular endothelial growth factor demonstrate significantly higher rates of progression than those with low expression.3 The sensitivities of nuclear matrix protein in urine for recurrence of superficial and invasive tumors are 71% and 100%, respectively.4 We have previously reported that the ratio of serum matrix metalloproteinase-2 to tissue inhibitors of metalloproteinase-2 is a useful predictor of postoperative recurrence of urothelial cancer.5 There are no useful serum markers, however, for predicting disease extension of bladder cancer or the overall survival of such patients. Therefore, the uncovering of new prognostic serum markers for determining disease extent and disease-free and overall survival of patients with bladder cancer has long been aspired to. Such markers would be conducive to the selection of patients who require adjuvant therapy after resection. The hepatocyte growth factor (HGF) stimulates cell motility and epithelial morphogenesis.6-9 This growth factor also induces proliferation of vascular endothelial cells through a transmembrane tyrosine kinase receptor encoded by a proto-oncogene, c-met, which leads to induction of angiogenesis.9-11 Moreover, levels of HGF in urine and tissue of muscle-invasive bladder cancer have been found to be higher than those in superficial bladder cancer.11,12 The serum levels of HGF in bladder cancer patients, however, have not been determined, and the relationship between serum HGF levels and the extent of bladder cancer and that between serum HGF level and tissue HGF level are not known. In the present study, we examined the relationship between the levels of HGF in serum and tissue of bladder cancer patients and evaluated the efficacy of serum HGF levels in predicting patient survival and the extent of transitional-cell carcinoma of the urinary bladder.
Patient Characteristics and Serum Collection Serum was collected between May 1985 and December 1997 at Hyogo Medical Center for Adults from 113 patients (aged 49 to 89 years; median age, 61; 84 men and 29 women) with transitional-cell carcinoma of the urinary bladder before treatment and from 200 healthy controls (aged 42 to 78 years; median age, 58 years; 90 men and 110 women). The serum samples were stored at -20°C until assayed. Before collection of serum from patients and healthy controls, their informed consent for the use of the samples in future experimentation was obtained. Details of the bladder cancer patients are summarized in Table 1.. Tumor stage and tumor differentiation were determined by the International Union Against Cancer–tumor-node-metastasis system and the World Health Organization classification, respectively. Of the 59 patients with superficial bladder cancer (  pT1), three had regional lymph node metastases. Of the 54 patients with muscle-invasive cancer ( pT2), 31 had no metastasis (pT2, 14 patients; pT3, 17) and 23 had metastasis (pT2, one patient; pT3, 13; pT4, seven; pTx, two). Of these 23 patients, lymph node metastasis alone was found in eight and visceral metastasis with or without lymph node metastasis in 15 (pulmonary metastasis alone in five patients, pulmonary and lymph node metastases in five, pulmonary, bone, and lymph node metastases in five). All bladder cancer patients who enrolled onto the present study exhibited no clinically malignant disease other than bladder cancer.
Tissue Collection Thirteen bladder cancer tissues were obtained at total cystectomy or endoscopic transurethral resection between July 1997 and December 1997 after obtaining patient consent for the use of the samples in future experimentation. Tumors were at stages pTa (four patients), pT1 (four), pT2 (one), pT3 (two), and pT4 (two) and were at grade 1 (one), grade 2 (five), and grade 3 (seven). Lymph node metastasis was detected in two patients, and both visceral and lymph node metastases were detected in two others. All tissues were immediately frozen in liquid nitrogen and stored at -80°C until use.
Enzyme Immunoassay The amounts of HGF in bladder cancer tissue were determined by a modified method of Rosen et al12 In brief, to prepare extracts, the tissues were thawed, washed, cut into small pieces, and homogenized in tissue extraction buffer (Mg2+, Ca2+ free Doulbeco’s PBS, 0.1 mmol/L phenylmethylsulfonyl fluoride). The homogenized tissue was sonicated in an ice water bath and then clarified by microcentrifuging. The extracts were assayed for HGF by enzyme immunoassay and for protein by a Bio-Rad protein assay kid (Bio-Rad Laboratories). The correlation between serum HGF levels and amounts of HGF in the tissues was determined by Pearson’s correlation coefficient.
Follow-Up Protocol for Recurrence in Patients With Superficial and Invasive Cancer After Surgery
Relationship Between Serum HGF and Patient Clinical Course
Disease-Free and Overall Survival of Patients With Multistage Bladder Cancer
The disease-free and overall survival rates of 15 patients with visceral metastasis and seven patients with pT4 disease according to their serum HGF levels, however, could not be statistically analyzed, because only one patient had visceral metastasis and a normal serum HGF level; the remaining patients showed elevated serum HGF levels. Moreover, patients with extensively invasive bladder cancer (T4 or
Statistics
Serum Levels of HGF Serum HGF levels are listed in Table 1. The mean ± SD value in normal healthy controls was 0.19 ± 0.05 ng/mL (range, 0.08 to 0.38 ng/mL). There was no significant difference in levels between male and female subjects of any age group (data not shown). In patients with superficial bladder cancer (mean ± SD, 0.29 ± 0.10 ng/mL), the levels were significantly higher than those in healthy controls (P = .001). In patients with muscle invasion, they were far higher than those in patients with superficial bladder cancer (P < .0001). The levels differed significantly among patients with tumors of different grades (P < .0003); the average level in patients with grade 3 tumors was higher than in patients with tumors of grade 1 or 2. In patients with vascular or lymphatic involvement, they were also much higher than those in patients without such involvement (P < .0001). Moreover, in patients with lymph node or visceral metastasis, the levels were significantly higher than those in patients without metastasis (P < .0001). In particular, patients with multiple lung metastases had higher levels than patients with metastases other than lung (median, 0.50 ng/mL; range, 0.30 to 1.23 ng/mL).
Statistical Determination of Elevation of Serum HGF
Amounts of HGF in Bladder Cancer Tissues
Relationship Between Serum HGF Levels and Patients’ Clinical Course The serum HGF levels in patients with muscle-invasive bladder cancer without metastasis, measured before and after total cystectomy in all seven patients, decreased after cystectomy (Fig 2A). The relationship between serum HGF level and recurrence is shown in Fig 2B. The site of recurrence was the lung alone in three patients, local recurrence in one, lymph node in 2, and lung, lymph node, and bone in three. The serum HGF level in eight of nine patients increased in association with disease recurrence; it showed no increase in the remaining one patient with lymph node recurrence.
Recurrence-Free and Overall Survival of Superficial Bladder Cancer According to Serum HGF Levels The disease-free and overall survival of 56 patients with superficial bladder cancer without lymph node metastasis according to serum HGF levels are shown in Fig 3. Among patients with high serum HGF levels ( 0.39 ng/mL), the 1- and 3-year disease-free survival rates were 100% and 55%, respectively (Fig 3A); among those with low serum HGF levels (< 0.39 ng/mL), on the other hand, they were 92% and 70%, respectively. There was no significant difference in disease-free survival between patients with low and those with high serum HGF levels (P = .674). Of five cases with high serum HGF level, two patients (40%) died of bladder cancer at 17 and 24 months after surgery. Of 51 patients with low serum HGF levels, however, only three (4.8%) died of myocardial infarction 5, 63, and 90 months after surgery with no evidence of disease. The 1- and 3-year overall survival rates of patients with low serum HGF levels were significantly higher than those of patients with high serum HGF levels (98% v 100% and 98% v 55%, respectively; P = .005) (Fig 3B).
Disease-Free and Overall Survival of Minimally Invasive Bladder Cancer Patients Who Underwent Curative Resection The 1- and 3-year disease-free survival rates of the 27 patients with high levels of serum HGF ( 0.39 ng/mL) were significantly lower (62.0% and 29.8%, respectively) than those of 13 patients with low levels of serum HGF (< 0.39 ng/mL; 100% and 89.0%, respectively; P < .001) (Fig 4A). The overall survival rates of the 27 patients were significantly lower than those of the 13 patients (P = .0028); the 1- and 3-year overall survival rates were 75.0% and 31.0%, respectively, for patients with high levels of serum HGF and 100% and 89.9%, respectively, for patients with low levels (Fig 4B).
Univariate and Multivariate Analyses of Disease-Free and Overall Survival in Superficial and Minimally Invasive Bladder Cancer Patients Who Underwent Curative Resection The serum HGF level was not a significant prognostic factor for disease-free survival of patients with superficial bladder cancer that involved pT stage, grade, or lymphatic involvement (Table 3). It was, however, a significant prognostic factor for only overall survival in patients with superficial bladder cancer on univariate analysis (P = .0326) (Table 3).
On the other hand, univariate analysis demonstrated that serum HGF level (P = .0091 and P = .0149), pT stage (P = .0163 and P = .0245), and vascular involvement (P = .0140 and P = .0170) were all significant prognostic factors for disease-free and overall survival, respectively, in minimally invasive bladder cancer (Table 4). Only the serum HGF level (P = .0162) was found to be a significant independent factor of disease-free survival on multivariate analysis. On the other hand, the serum HGF level (P = .0282) and vascular involvement (P = .0497) were found to be significant independent prognostic factors for overall survival on multivariate analysis.
Metastatic lesions are established in multiple steps.14 During these steps, several growth factors and cytokines regulate tumor invasion, motility, and angiogenesis via extracellular matrix degradative enzymes.15,16 HGF also stimulates invasion of tumor cells in vitro and induces angiogenesis in vivo.6-10 Breast cancer patients with a high HGF content in cancer tissue have significantly shorter relapse-free and overall survival than patients with a low HGF content.17 Lung cancer patients with higher levels of HGF in their tissue have poorer overall survival than patients with lower HGF levels.18 Also, multivariate analysis has demonstrated that tissue HGF level is a strong independently negative prognostic factor for patients with non–small-cell lung cancer, which suggests that the HGF level in cancer tissue could be a useful indicator of risk of relapse and death in patients with early-stage lung cancer.18 Furthermore, elevated serum HGF level has been found in patients with other types of neoplasia, including gastric cancer and multiple myeloma, which suggests that it is associated with disease progression.19,20 Circulating levels of HGF are elevated in one third of patients with gastric cancer, particularly in those with high grades of histologic tumor invasion and metastasis.19 Taniguchi et al19 have therefore postulated that HGF might play an important role in the progression of gastric cancer. The serum HGF level is elevated in 43% of patients with multiple myeloma, evincing poor prognosis; therefore, serum HGF level could also be a useful prognostic factor of multiple myeloma.20 In the present study, serum HGF levels in more advanced bladder cancer were significantly higher than those in low-stage cancer; in particular, the levels in patients whose cancer had metastasized were significantly higher than those in patients whose tumor had not metastasized. The levels decreased after cystectomy and increased with recurrence. Therefore, the major source of elevated serum HGF level could be the tumor itself. The amounts of HGF in muscle-invasive, high-grade or lymph node metastatic bladder cancer tissues were significantly higher than those in superficial, low-grade or nonmetastatic tissue, and serum HGF levels correlated well with the amounts of tissue HGF. Thus, our results indicate that serum HGF titers tend to reflect disease activity and that particularly high serum and tissue titers of HGF are found in muscle-invasive or metastatic cancers.
Overall survival of patients with superficial bladder cancer with low serum HGF levels was significantly higher than that of patients with high levels; however, the disease-free survival was not significantly different between the two groups. The serum HGF level was a significant prognostic factor for overall survival in patients with superficial bladder cancer. Moreover, in cases of minimally invasive bladder cancer in which curative resection was carried out, the prognosis of patients with high levels of serum HGF was significantly more unfavorable than that of patients with low levels. The traditional prognostic factors for bladder cancer are pT, tumor grade, and metastasis.21 Multivariate analysis in our study, however, demonstrated that the serum HGF level was a significant prognostic factor for both disease-free and overall survival of patients who had minimally invasive bladder cancer and who underwent curative resection. On the other hand, pT, tumor grade, and lymph node metastasis seemed not to be significant as prognostic factors. The reason for the discrepancy between our observations and those of previous reports is unclear; however, a possible explanation is that only minimally invasive bladder cancers were used in our multivariate analysis, whereas patients with both extensive invasion and extensive lymph node metastasis (T4 or HGF, occasionally detected in various tissues, is exclusively in the inactive single-chain pro-HGF form, and native HGF is secreted by cells as a pro-HGF form; moreover, processing pro-HGF to form active heterodimers is necessary for HGF to fully activate the HGF receptor and to induce biologic responses in target cells.22 Three converting enzymes have been described in serum and tissues, including blood coagulation factor XIIa, HGF activator, and urokinase-type plasminogen activator (uPA).22,23 In human bladder cancer tissue, the uPA level correlates well with tumor invasive and metastatic potential, the uPA content of high-grade transitional-cell carcinoma of the bladder is significantly higher than that of low-grade cancer, and the level of uPA in tumor tissue seems to be a significant prognostic factor.24 These findings suggest that both uPA and pro-HGF may be highly expressed in high-grade and advanced bladder tumors and that the serum level of HGF is, therefore, elevated in patients with such tumors, which results in tumor progression and poor prognosis. Moreover, HGF is a growth factor that shows biologic activities through a receptor (c-met); bladder cancer tissue with great amounts of HGF also shows high expression of c-met.11,25 On the other hand, the expression of c-met is found more often in metastatic prostatic carcinoma than in nonmetastatic tumors.26 HGF and c-met may operate in a paracrine manner in influencing prostatic carcinoma cell behavior, including cell proliferation and scattering, and c-met expression may be associated with prostatic carcinoma progression.26 Because the present study focused on determining serum HGF levels as a prognostic factor of bladder cancer, the expression of c-met in bladder cancer tissues was not examined. In conclusion, among all bladder cancer patients, those with metastatic bladder cancer had the highest levels of HGF in serum and tissues. The serum HGF levels correlated well with the amounts of tissue HGF. Patients with minimally invasive bladder cancer and high serum HGF levels had short disease-free and overall survival after curative resection. Moreover, the overall survival of patients with superficial bladder cancer and high serum HGF was also short. These findings suggest that the HGF level in serum could be a predictor of patient survival and the extent of bladder cancer.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test; among patients with tumors of different grades, however, differences of serum HGF levels and the elevation were determined by analysis of variance. The disease-free and overall survival rates of patients with minimal locally-invasive bladder cancer, who underwent curative resection, were determined by the Kaplan-Meier method, and the difference was determined by the log-rank test. Disease-free survival was defined as the period between surgery and the detection of the first local recurrence or distant metastasis or the end of the study. Overall survival was defined as the period from surgery to the patient’s death. Factors related to disease-free and overall survival in patients who underwent curative resection were analyzed by Cox’s proportional hazards regression model.
