Hiroki Yagasaki md, Department of Urology, Nihon University School of Medicine, 30-1 Oyaguchi Kamichou, Itabashi-ku, Tokyo 173-0032, Japan. Email: firstname.lastname@example.org
Aim: The present study was carried out to clarify whether a histopathological analysis of vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase 2 (MMP-2) can help predict the outcome of renal cell carcinoma (RCC). We examined the expression of VEGF, TGF-β1 and MMP-2 in a large series of RCC with a long follow-up, based on histopathological factors and survival.
Methods: Immunostaining for VEGF, TGF-β1 and MMP-2 was performed on formalin-fixed, paraffin-embedded tissue sections from 84 patients with RCC who underwent nephrectomy at our institution between 1985 to 2000. The microvessel density (MVD) of tumor tissue was measured after it immunohistochemically stained with CD105 (Endoglin) monoclonal antibody.
Results: A significant association was observed in the expression of VEGF and TGF-β1 regarding the stage (P < 0.01, P < 0.01), nuclear grade (P < 0.01, P < 0.01) and MVD (P < 0.001, P < 0.001), respectively. However, no correlation was found among the results of MMP-2, nuclear grade and MVD. A multivariate analysis demonstrated both the nuclear grade and MVD to be independent prognostic factors.
Conclusion: Our results suggested that the expression of both VEGF and/or TGF-β1 can be useful predictive prognostic factors RCC. In addition, a multivariate analysis demonstrated MVD to be an independent prognostic factor of RCC.
Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney, and accounts for approximately 3% of all adult malignancies.1 Due to the increased use of imaging procedures during routine medical check-ups for other diseases, the incidental detection of RCC has been increasing recently. RCC is now the 10th leading cause of cancer mortality in Japan, with more than 2 to 3 new cases per 100 000 annually and 3000 deaths reported in 1997.1
The incidence of metastatic disease among patients with newly diagnosed RCC is approximately 30%, and the prognosis is poor in most cases. In addition, 40% of all individuals undergoing a surgical resection will eventually develop metastasis.
Clinical and experimental studies have shown that angiogenesis is essential for both tumor growth and metastasis. Microvessel density (MVD) has been shown to be correlated with an advanced pathological stage and poor clinical outcome in various cancers, including breast, prostate and cervical cancers.2–4 The process of angiogenesis consists of various stages, including the detachment of the endothelium, resolution of the basement membrane and extracellular matrix (ECM), proliferation of the endothelium and new vessel formation with a new basement membrane. However, exactly how the angiogenic factors are involved in this process has not yet been completely elucidated.5
Due to the fact that RCC is one of the most highly vascularized solid malignancies, RCC angiogenesis may thus play a role in several steps of invasion. The expression level of several genes, vascular endothelial growth factor (VEGF), extracellular matrix metalloproteinases (MMPs) and transforming growth factor (TGF-) β have all been studied independently and shown to correlate with the prognostic factors of RCC patients.6–9 However, previous studies have not shown any correlation among these factors.
The aim of this study was to closely investigate the expression of these factors, elucidate how they are related to the process of angiogenesis including VEGF, TGF-β1 and MMP-2 in RCCs and assess these factors as prognostic factors with reference to MVD in tumor tissue.
Formalin-fixed, paraffin-embedded tissue samples were obtained from 84 consecutive patients who underwent nephrectomy for primary RCC without neoadjuvant therapy in the Department of Urology, Surugadai Nihon University Hospital, Japan, between 1985 and 2000.
The clinicopathological factors were evaluated based on the criteria according to the classification of RCC of the Union Internationale Contre Le Cancer and the American Joint Committee on Cancer.10–12 The mean age of patients was 60 years, and the mean size of tumors was 50 mm. Forty-eight cases were classified as stage I, 12 cases were stage II, 18 cases were stage II and 6 cases were stage IV. According to the histological grading, 38 cases were grade 1, 29 cases were grade 2 and 17 cases were grade 3.
To analyze the immunohistochemical expression of VEGF, TGF-β1 and MMP-2, 5 µm serial sections were cut from a representative block of formalin-fixed, paraffin-embedded RCC tissue. The sections were deparaffined with xylene and then dehydrated in a series of graded ethanol. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxidase in absolute methanol for 30 min at room temperature. Anti-VEGF rabbit polyclonal antibody, anti TGF-β1 rabbit polyclonal antibody and anti-MMP-2 goat polyclonal antibody (1 : 100, all supplied by Santa Cruz Biotechnology, Santa Cruz, California, USA), were used as a primary antibody with a standard avidin–biotin immunoperoxidase method at 4°C for 18 h incubation. The tissue-bound peroxidase was visualized by incubating the sections with 0.02% 3–3′diaminobenzidine in phosphate buffer (pH 7.2) containing 0.03% hydrogen peroxide for 5 min. The sections were lightly counter-stained with Mayer's hematoxylin. The staining intensity of VEGF, TGF-β1 and MMP-2 was classified into a four-grade scale: 0, absence of immunostaining or faint membranous staining of rare tumor cells; 1+, membranous staining in most tumor cells; 2+, diffuse membranous and/or cytoplasmic staining in groups of tumor cells; and 3+, significant cytoplasmic staining in most tumor cells (Fig. 1).
Vascularization was demonstrated by an immunohistochemical analysis for CD105 (DAKO, Carpinteria, California, USA) using a catalyzed signal amplification system (DAKO), based on the peroxidase–catalyzed deposition of a biotinylated phenol compound tyramide. The MVD was evaluated under light microscopy according to the procedure of Weidner et al.2 The mean MVD was obtained by counting each vessel identified within an aggregate of 30 consecutive high power fields (HPF) (× 400).
The data were analyzed using the Statview J 5.0 software package (SAS Institute, Cary, NC, USA). For a statistical analysis, the nuclear grade was divided into two groups (1, and 2 or 3) as was the clinical stage (I or II, and III or IV). Fisher's exact test was used to analyze the relationships among the results of immunohistochemistry of VEGF, TGF-β1, MMP-2 and other pathological features. The correlation between the expression of VEGF, TGF-β1and MMP-2 and MVD was determined using the Kruskal–Wallis test. Life tables were drawn by the Kaplan–Meier method, and the survival curves were compared using the log–rank test. Cox multiple regression analysis was performed to determine any independent predictive values. P < 0.05 was considered to be statistically significant.
Immunohistochemistry of VEGF, TGF-β1 and MMP-2
Regarding the immunohistochemistry of VEGF, 17 cases were 0 (20.2%), 27 cases were 1+ (32.2%), 21 cases were 2+ (25.0%) and 19 cases were 3+ (22.6%) (Fig. 1). For TGF-β1, 7 cases were 0 (8.3%), 31 cases were 1+ (36.9%), 25 cases were 2+ (29.8%) and 21 cases were 3+ (25.0%) (Fig. 2). MMP-2 staining was 0 in 12 cases (14.3%), 1+ in 26 cases (30.9%), 2+ in 36 cases (42.9%) and 3+ in 10 cases (11.9%) (Fig. 2). The immunohistochemical expression of each protein and their correlations with tumor size, histological stage and nuclear grade are summarized in Table 1. A significant association was observed in the expression of VEGF and TGF-β1 with the clinical stage (P < 0.01, P < 0.01) and nuclear grade (P < 0.01, P < 0.01). Both the clinical stage and tumor size showed a significant association with the expression of MMP-2 (P < 0.05, P < 0.05). However, no correlation was found between MMP-2 and nuclear grade.
Table 1. Expression of vascular endothelial-derived growth factor (VEGF), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-2 (MMP-2) according to tumor size, stage and nuclear grade
The mean MVD value was 17.99 ± 11.94. Based on our criterion, both the nuclear grade and clinical stage demonstrated a significant association with MVD (nuclear grade 1 versus 2 or 3, clinical stage I or II versus III or IV, 20.60 ± 11.22 versus 15.79 ± 12.20; P < 0.01, 20.45 ± 12.11 versus 12.46 ± 9.46; P < 0.01, respectively). The expression of VEGF, TGF-β1 and MMP-2 by MVD are summarized in Table 2. A significant association was seen in the expression of VEGF and TGF-β1 with MVD (P < 0.001, P < 0.001).
Table 2. Expression of vascular endothelial-derived growth factor (VEGF), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-2 (MMP-2) according to microvessel density (MVD)
The median follow-up period of the 84 patients was 33 months ranging from 1 month to 154 months. Eight patients died during the follow-up period. To clarify MVD potential impact on survival, the patients were divided two groups according to the median MVD expressed as microvessels per HPF (16.85). The log–rank test showed the MVD (Fig. 3) and expression of VEGF and TGF-β1 were well correlated with survival. However, the expression of MMP-2 was not correlated with survival (data not shown).
The individual prognostic significance of the clinicopathological findings and expression of angiogenic factors was observed by univariate and multivariate analyzes (Table 3). Both the nuclear grade and MVD were found to be significant independent prognostic factors.
Table 3. Relative protein expression of angiogenic factors (n = 84)
Angiogenesis is well known to be one of the most important factors for tumor progression in malignant tumors. It is also known to be regulated by the interaction between the promoter of angiogenesis and its inhibitor. During the process of tumor progression and subsequent metastasis, tumor micro-environment, particularly angiogenesis play a key role in both establishing a neocapillary network and local invasion to the host stroma.13
VEGF has been shown to play an important role in vascular biology of tumor growth, particularly as a mediator of angiogenesis. Previous studies have demonstrated the expression levels of VEGF mRNA in RCC to be higher than that of normal kidney tissue based on a northern blot analysis14 and in situ hybridization.15 Paradis et al. also observed VEGF located in neocapillaries of RCC using immunohistochemical study.6 They explained that intratumoral blood vessels express VEGF receptor's mRNA and then VEGF released by tumor cells binds to VEGF receptor on endothelial cells. In contrast, the expression of VEGF mRNA is not always well correlated with the expression of VEGF protein.16 In the present study, the immunohistochemical expression of VEGF was assessed semiquantitatively using a four-grade scale. VEGF is commonly expressed in cancer cells while the expression status is significantly related to both the high clinical stage and high nuclear grade of RCC (P < 0.01). These results suggest that VEGF can be a useful predictive factor for both tumor growth and prognosis.
TGF-β belongs to a family of morphogenetic growth factors which is generally known to regulate the growth, differentiation and immunological function of the cancer cell environment.17 In addition, TGF-β also has discrepant potencies, namely as a potent growth inhibitor and, simultaneously, as a growth stimulator.18 This discrepancy might be related to the immunosuppression by inducing extracellular deposition and decreasing the amount of basement membrane protein. Although TGF-β has been indicated to be one of the potent growth inhibitor regulators for epithelial cells,18 our results do not fully support this idea.18 In addition, recent studies have demonstrated that an over expression of TGF-β is associated with tumor progression in several cancers.18 In this study, the expression of TGF-β was shown to be associated with both of the high clinical stage and the high nuclear grade (P < 0.01).
The expression of various types of MMPs has been demonstrated in malignant tumors. MMP-2 degrade type IV collagen is a major component of basement membrane and is known to be closely related to cancer cell invasion and metastasis.19,20 The process of proteolysis of the basement membrane and extracellular matrix components is thought to be an essential step in tumor invasion and metastasis. In the process neovascularization localized degradation of capillary basement membranes is required as an early event. During tube formation by human umbilical vein endothelial cells grown on matrigel has been shown to require MMP-2 activity.21 The deleted MMP-2 activity has also been shown to decrease tumor-induced angiogenesis in an experimental study of metastasis in mice.21
Kugler et al. demonstrated an overexpression of mRNA of MMP-2, MMP-9 and MMP-14 in tumor tissue compared with their non-tumorous tissue, and an elevated activity of MMP-2 in the advanced cancers of RCC.9 Lein et al. also reported the protein expression of MMP-9 in cancer tissue to be significantly higher than that of normal tissue, while there was no significant difference in the expression of MMP-2 between the cancer and normal tissue.22 These different results can be partly explained due to the difference of grade and clinical stage of the samples examined. In addition, the semiquantitative measurement of mRNA of MMPs by RT–PCR is highly sensitive, but does not show protein expression and its enzymatic activity. In the present study, the expression of MMP-2 positively correlated with both the tumor size and stage (P < 0.05). These results suggest that MMP-2 might thus be associated with tumor growth and progression.
An increased MVD has been reported to be associated with an early onset of tumor progression in various types of malignant neoplasms, including breast, bladder and prostate cancers.3,23,24 Although a positive correlation was observed between increased MVD and a good prognostic outcome in RCC, most studies have shown an increased MVD to be related to a poor prognosis. In the present study, we used CD105 as a marker to visualize microvessels. CD105 is known to be a type III receptor of TGF-β molecule and to be up-regulated in tumors.25 Brewer et al. suggested that CD105 could preferentially detect vessels undergoing neo-angiogenesis and the staining is very selective for vessel endothelium.4 Anti-CD105 antibody reacts specifically with angiogenic endothelial cells, and the results using this antibody tend to produce less false–positive staining than other commonly used antibodies against vascular endothelial cells such as anti-CD31 and antifactor VIII related antigen.24 Our results also support the hypothesis that a low level of MVD is related to a poor prognosis in RCC. These results can be explained by the fact that a decreased MVD is associated with tumor fibrosis and the development of large vascular channels.26,27
As mentioned earlier, MVD is related to the expression of VEGF. Hemmerlein et al. observed a decreased VEGF mRNA expression in RCC with a high proliferative activity.28 At an early stage, tumor formation is known to be accompanied by a high MVD. At this stage, highly proliferating tumors induce a high angiogenetic activity. This belief might be supported by the finding that the smaller tumors show a higher MVD than larger ones. A high proliferative activity of tumor cells results in a faster increase in tumor size. When tumor tissue is exposed to hypoxic condition, the level of VEGF could increase. The rapid increase in tumor volume may be accompanied with a reduced MVD. Generally, rapid tumor growth induces a hypoxic condition in tumor tissue. A change in the micro-environment in the tumor tissue may lead to a decrease in tumor proliferation, thus resulting in an overexpression of VEGF mRNA.28
In addition, basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α) are also well known to play an important role in angiogenesis,7 and in this study, MVD was found to correlate with the expression of TGF-β1. Although the relationship between MVD and TGF-β has not yet been fully investigated, our results indicated that TGF-β may play an important role in the process of angiogenesis in RCC. The endothelial expression of MMP-2 indicates the activation of angiogenesis in RCC, the expression of which can be induced by VEGF. Hemmerlein et al. described that the expression of endothelial MMPs and microvessels without any contribution of pericytes is more often seen in cancer tissue than in non-cancer tissue.28 Regarding the relationship between MMPs and VEGF expression, a significant correlation was reported between VEGF expression and MMP-2 immunostaining in ovarian cancer,29 and also between VEGF immunostaining and activated MMP-2 on zymography in breast cancer,30 thus suggesting an interesting link between neoplastic invasiveness and neo-angiogenesis. In this study, no significant correlation was observed between the MVD and MMP-2.
A multivariate analysis of the results showed a low MVD finding to be a stronger predictor for survival than the nuclear grade. The expressions of VEGF, TGF-β1 and MMP-2 are well known to be associated with MVD. As a result, these factors are considered to be useful predictors in the diagnosis of RCC.
In addition to the expression status of VEGF and TGF-β, a multivariate analysis showed MVD to be an independent predictive factor for the prognosis of RCC.