Association of hypoxia-inducible factors 1alpha and 2alpha with activated angiogenic pathways and prognosis in patients with endometrial carcinoma

BACKGROUND: Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha are essential regulatory proteins for the adaptation of tumor cells to hypoxia, and they stimulate angiogenesis through activation of the vascular endothelial growth factor (VEGF) gene. METHODS: HIF-1alpha and HIF-2alpha proteins were studied immunohistochemically in a group of 81 patients with Stage I endometrial adenocarcinoma of the endometrioid cell type. The results were correlated with intratumoral angiogenesis, the expression of the angiogenic factors VEGF and thymidine phosphorylase (TP), and the VEGF/receptor (VEGF/KDR) complex. Relations also were sought with estrogen receptor (ER) and progesterone receptor (PR), with the apoptosis-related proteins bcl-2 and p53, with several histopathologic parameters, and with patient prognosis. In addition, a sample of 25 normal endometria at various phases of the menstrual cycle was studied for the presence of HIF-1alpha and HIF-2alpha. RESULTS: HIF-1alpha expression was detected in 49% of endometrial carcinomas. The expression was cytoplasmic or mixed nuclear/cytoplasmic. HIF-1alpha expression was associated with up-regulation of the VEGF pathway and with increased standard microvessel density (sMVD) and activated VEGF/KDR microvessel density (aMVD). It also was associated with a poor prognosis in both univariate and multivariate analyses. HIF-2alpha protein showed a pattern of expression similar to the pattern seen in HIF-1alpha, but expression of HIF-2alpha protein occurred in only 17% of endometrial carcinomas, and it was associated with increased TP reactivity. There also was a relation of HIF-1alpha expression with well-differentiated endometrial neoplasms, and there was a marginal association of HIF-1alpha and HIF-2alpha with ER expression. With reference to normally cycling tissues, HIF-1alpha nuclear/cytoplasmic expression was particularly strong in the samples of early proliferative phase endometrium compared with HIF-2alpha protein expression, which showed a constant reaction throughout the menstrual cycle. CONCLUSIONS: The up-regulation of HIF-1alpha and, to a lesser extent, of HIF-2alpha is a common event in Stage I endometrial adenocarcinomas. In these tumors, HIF-1alpha expression is related to increased angiogenesis, through activation of the VEGF angiogenic pathway, and to an unfavorable prognosis. HIF-2alpha accumulation is associated with increased expression of the angiogenic factor TP.     

Vascular phenotype in angiogenic and non-angiogenic lung non-small cell carcinomas

We have previously described a group of non-small cell lung carcinomas without morphological evidence of neo-angiogenesis. In these tumours neoplastic cells fill up the alveoli and the only vessels present appear to belong to the trapped alveolar septa. In the present study we have characterised the phenotype of the vessels present in these non-angiogenic tumours, in normal lung and in angiogenic non-small cell lung carcinomas. The vessels, identified by the expression of CD31, were scored as mature when expressing the epitope LH39 in the basal membrane and as newly formed when expressing alphaVbeta3 on the endothelial cells and/or lacking LH39 expression. In the nine putative non-angiogenic cases examined, the vascular phenotype of all the vessels was the same as that of alveolar vessels in normal lung: LH39 positive and alphaVbeta3 variable or negative. Instead in 104 angiogenic tumours examined, only a minority of vessels (mean 13.1%; range 0--60%) expressed LH39, while alphaVbeta3 (in 45 cases) was strongly expressed on many vessels (mean 55.5%; range 5--90%). We conclude that in putative non-angiogenic tumours the vascular phenotype is that of normal vessels and there is no neo-angiogenesis. This type of cancer may be resistant to some anti-angiogenic therapy and different strategies need to be developed.     

Coexpression of hypoxia-inducible factors 1alpha and 2alpha, carbonic anhydrase IX, and vascular endothelial growth factor in nasopharyngeal carcinoma and relationship to survival.

PURPOSE: Tumor hypoxia is known to be associated with resistance to chemotherapy, radiotherapy, and poorer survival. Recently, it is shown that hypoxia induces the expression of hypoxia-inducible factor-1alpha and 2alpha (HIF-1alpha and HIF-2alpha), which then up-regulates the expression of downstream genes such as carbonic anhydrase IX (CA IX) and vascular endothelial growth factor (VEGF). EXPERIMENTAL DESIGN: We examined the expression of HIF-1alpha, HIF-2alpha, CA IX, and VEGF by immunohistochemistry in nasopharyngeal carcinoma (NPC) biopsies from 90 consecutive patients recruited between 1994 and 1997 in a randomized controlled trial of chemoradiation in locally advanced NPC and investigated their relationship with survival. RESULTS: HIF-1alpha was expressed in 52 of 90 (58%), HIF-2alpha in 6 of 89 (7%), CA IX in 51 of 90 (57%), and VEGF in 54 of 90 (60%) of tumors. Tumor HIF-1alpha expression correlated significantly with that of CA IX (P = 0.008) and VEGF (P = 0.003). High tumor HIF-1alpha expression was associated with a trend for poor overall survival (P = 0.06). Tumors with a positive hypoxic profile (defined as high expression of both HIF-1alpha and CA9) were associated with worse progression-free survival (P = 0.04). Tumors with both hypoxic and angiogenic profile (defined as high VEGF expression) were associated with a worse progression-free survival (P = 0.0095). CONCLUSION: Overexpression of HIF-1alpha, CA IX, and VEGF is common in NPC, which is probably related to hypoxia up-regulated expression involving a HIF-dependent pathway, and is associated with poor prognosis. Targeting the hypoxia pathway may be useful in the treatment of NPC.     

Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer

Vascular endothelial growth factor (VEGF) is an important angiogenic factor, linked to poor outcome in human malignancies including non-small cell lung carcinoma (NSCLC). We used the 11B5 monoclonal antibody recognizing the VEGF/KDR complex (R. A. Brekken et al., Cancer Res., 58: 1952-1959, 1998) to assess the VEGF expression in cancer cells and the VEGF/KDR activated microvessel density (aMVD) in early operable NSCLC. The JC70 anti-CD31 monoclonal antibody was used to assess the standard MVD (sMVD). The aMVD was significantly higher in the invading front of the tumors and in the normal lung adjacent to the tumors as compared with normal lung distant to the tumor or to inner tumor areas (P < 0.0002). The sMVD was higher in the normal lung and decreased from the invading front to inner tumor areas (P < 0.0001). However, the vascular activation (aMVD:sMVD) was 4-6 times higher in the tumor areas as compared with lung from normal individuals (36-58% versus 9%; P < 0.0001). Fibroblast 11B5 reactivity, noted in 25% of cases, correlated with high aMVD and sMVD in the inner tumor areas. Multivariate analysis showed that aMVD was the most potent and independent prognostic factor (P = 0.001; t-ratio, 3.28). It is concluded that intense VEGF/KDR angiogenic pathway activation is a tumor-specific feature in more than 50% of NSCLC cases and is associated with poor postoperative outcome. Clinical trials involving targeting of the VEGF/KDR-positive vasculature with specific antibodies, such as 11B5, are, therefore, encouraged.     

Angiogenic interactions of vascular endothelial growth factor, of thymidine phosphorylase, and of p53 protein expression in locally advanced gastric cancer

The assessment of the angiogenic profile of tumors may become an important tool as a guide for the inclusion of novel drugs and molecular therapies into the standard chemoradiotherapy policy. Several studies have shown the prognostic importance of microvessel density (MVD) and of angiogenic factor expression in operable gastric cancer. In the present study we investigated, with immunohistochemistry the MVD, the expression of vascular endothelial growth factor (VEGF) and of thymidine phosphorylase (TP) expression, as well as the nuclear expression of p53 protein, in a series of patients with locally advanced inoperable gastric cancer. A strong association of VEGF with TP expression was noted (P = 0.005), and tumors coexpressing these factors had a statistically higher MVD (P = 0.0001). Nuclear p53 accumulation was also related to a high MVD (P = 0.004), and this was independent of VEGF or TP expression. Microvessel density showed a bell-shaped association with prognosis; cases with an intermediate MVD exhibit a favorable outcome (P < 0.05). A trend of nuclear TP expression to define a group of patients with poorer prognosis was noted (P = 0.06), while none of the remaining variables showed any significant association. The immunostaining results allowed the grouping of the angiogenic profile in four major categories: 1) highly vascularized tumors with VEGF and/or TP expression (about 36% of cases); 2) highly angiogenic tumors with p53 nuclear accumulation and low VEGF/TP expression (7% of cases); 3) poorly vascularized tumor with low VEGF/TP and negative nuclear p53 staining (32% of cases); 4) poorly vascularized tumors with TP expression (7% of cases). Specific therapies targeting hypoxia, VEGF, or TP expression as well as p53 gene therapy have entered clinical experimentation or are already available for clinical use. Using the suggested markers more than 80% of locally advanced gastric carcinomas can be grouped in different categories according to their angiogenic profile. Such a categorization may be useful for phase III trials on novel therapies targeting the major angiogenesis-related features studied here.     

The angiogenic receptor KDR is widely distributed in human tissues and tumours and relocates intracellularly on phosphorylation. An immunohistochemical study

AIMS: Angiogenesis is an important factor in tumour growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) or KDR plays a crucial role in angiogenesis. The aim of this study was to raise and characterize antibodies against phosphorylated KDR which could be used for studies on human tissues to assess KDR activation and novel inhibitors of KDR activation in clinical trials. METHODS AND RESULTS: Three monoclonal antibodies and one rabbit polyclonal antiserum were produced. The specificity of the antibodies was confirmed by ELISA. One of the mouse antibodies and the rabbit polyclonal antiserum reacted with a 200-kDa band on a Western blot of human umbilical vein endothelial cell (HUVEC) lysates, the molecular weight of KDR. Immunohistochemical staining showed that phosphorylated KDR is present in a wide variety of normal tissues including liver, colon and placenta, and is not restricted to endothelium. It was also present in a number of human tumours including breast carcinomas, colonic carcinomas and non-Hodgkin's lymphomas. The pattern of staining was membranous, cytoplasmic and nuclear. CONCLUSIONS: This study has shown that phosphorylated KDR is present in a wide variety of tumour and tissue types and is not confined to endothelium.