Tumor vascularity: a histological measure of angiogenesis and hypoxia.

In this study we sought to clarify the relationship between tumor vascularity, hypoxia, and angiogenesis in human cervix tumors. Two hypotheses were established: first, that measurement of tumor vascularity can provide a histological assessment of both hypoxia and angiogenesis; and second, that expression of angiogenesis-related proteins will provide a surrogate measure of tumor hypoxia. To test the first hypothesis, we studied the prognostic significance of tumor vascularity measured as both intercapillary distance (ICD; thought to reflect tumor oxygenation) and microvessel density (MVD; the hotspot method that provides a histological assessment of tumor angiogenesis). The relationship was also examined of tumor hypoxia, measured using an Eppendorf needle electrode [percentage of values less than 5 mm Hg (HP5)], with ICD and MVD. To test the second hypothesis we examined the relationship between HP5 and the expression of angiogenesis-associated proteins [vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF)]. All of the biological measurements were made on pretreatment tumors. Analysis of data was carried out using log-rank statistics, Cox multivariate analysis, and Spearman's rank correlation. Both ICD and MVD were significant independent prognostic factors for local control. Patients with poorly vascularized tumors (long ICD) had poor local control (P = 0.042). However, patients with poorly vascularized tumors, measured as low MVD, had good local control (P = 0.036). For 107 patients in whom both of the measurements were obtained on the same tumor sections, ICD and MVD provided independent prognostic information in multivariate analysis. There was a significant correlation between tumor hypoxia and ICD (P < 0.005) but not MVD (P = 0.41). There was no relationship between hypoxia and the expression of angiogenic factors (VEGF, PD-ECGF). These analyses show that measurement of tumor vascularity can provide different biological information that is dependent on the method used. It is, therefore, important that studies measuring vascularity should include an appropriate definition. There is no relationship between hypoxia and angiogenesis in advanced carcinoma of the cervix and examining the levels of angiogenic proteins may not have a role in assessing hypoxia in cervix cancer.

Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix.

There is increasing evidence that hypoxia-regulated gene expression influences tumor aggressiveness, contributing to the poorer outcome of patients with hypoxic tumors. The role of the transcriptional complex hypoxia-inducible factor-1 as an important mediator of hypoxia-regulated gene expression is one of the best documented pathways. Recently, it has emerged that certain tumor-associated carbonic anhydrases (CAs) can be added to the list of known hypoxia-inducible factor-responsive genes. Here we show that the immunohistochemical expression of the tumor-associated CA IX is correlated with the level of hypoxia in human cervical tumors. We performed a prospective study in 68 patients where needle electrodes were used to make direct measurements of tumor oxygenation levels. CA IX expression was evaluated immunohistochemically in pretreatment tumor biopsies. There was a significant positive correlation between the level of tumor hypoxia (HP5) and the extent of CA IX expression. A retrospective study of 130 squamous cell cervical carcinomas demonstrated that a semiquantitative immunohistochemical analysis of CA IX expression in tumor biopsies is a significant and independent prognostic indicator of overall survival and metastasis-free survival after radiation therapy. These studies provide clinical evidence that CA IX expression is up-regulated in hypoxic human cervical tumors and is associated with a poor prognosis. CA IX may act as an intrinsic marker of tumor hypoxia and poor outcome after radiation therapy. The level of CA IX expression may be used to aid in the selection of patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.

Tumour oxygenation levels correlate with dynamic contrast-enhanced magnetic resonance imaging parameters in carcinoma of the cervix.

BACKGROUND AND PURPOSE: The Eppendorf pO(2) histograph is the 'gold standard' method for measuring tumour oxygenation. The method is not suitable for widespread application because its use is limited to accessible tumours. A non-invasive imaging technique would be an attractive alternative. Therefore, the relationships between tumour oxygenation and dynamic contrast-enhanced magnetic resonance imaging (MRI) parameters were investigated. MATERIALS AND METHODS: The study comprised 30 patients with carcinoma of the cervix. Tumour oxygenation was measured pre-treatment as median pO(2) and the proportion of values less than 5 mmHg (HP5) using a pO(2) histograph. Repeat measurements were obtained for nine patients following 40-45 Gy external beam radiotherapy giving a total of 39 measurements. Dynamic contrast-enhanced MRI using gadolinium was performed prior to obtaining the oxygenation data. Time/signal intensity curves were generated to obtain two standard parameters: maximum enhancement over baseline (SI-I) and the rate of enhancement (SI-I/s). RESULTS: Using the 39 measurements, there was a significant correlation between SI-I and both median pO(2) (r=0.59; P<0.001) and HP5 (r=-0. 49; P=0.002). There was a weak, borderline significant correlation between SI-I/s and both median pO(2) (r=0.29; P=0.071) and HP5 (r=-0. 34; P=0.037). There was a significant relationship between tumour size and SI-I (r=0.54; P<0.001), but not SI-I/s. In 29 tumours, where data were available, there was no relationship between histological assessment of tumour angiogenesis (intra-tumour microvessel density; IMD) and either MRI parameter. CONCLUSIONS: Tumour oxygenation levels measured using a pO(2) histograph correlate with dynamic contrast-enhanced MRI parameters. Therefore, non-invasive dynamic MRI may be a method for measuring hypoxia in human tumours.

No relationship between thymidine phosphorylase (TP, PD-ECGF) expression and hypoxia in carcinoma of the cervix.

The expression of hypoxia-regulated genes promotes an aggressive tumour phenotype and is associated with an adverse cancer treatment outcome. Thymidine phosphorylase (TP) levels increase under hypoxia, but the protein has not been studied in association with hypoxia in human tumours. An investigation was made, therefore, of the relationship of tumour TP with hypoxia, the expression of other hypoxia-associated markers and clinical outcome. This retrospective study was carried out in patients with locally advanced cervical carcinoma who underwent radiotherapy. Protein expression was evaluated with immunohistochemistry. Hypoxia was measured using microelectrodes and the level of pimonidazole binding. There was no relationship of TP expression with tumour pO(2) (r=-0.091, P=0.59, n=87) or pimonidazole binding (r=0.13, P=0.45, n=38). There was no relationship between TP and HIF-1alpha, but there was a weak borderline significant relationship with HIF-2alpha expression. There were weak but significant correlations of TP with the expression of VEGF, CA IX and Glut-1. In 119 patients, the presence of TP expression predicted for disease-specific (P=0.032) and metastasis-free (P=0.050) survival. The results suggest that TP is not a surrogate marker of hypoxia, but is linked to the expression of hypoxia-associated genes and has weak prognostic power.

Vascular endothelial growth factor (VEGF) expression is a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix.

The aim of the study was to evaluate VEGF expression in tumour biopsies as a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix. A retrospective study was carried out on 100 patients. Pre-treatment tumour VEGF expression was examined immunohistochemically in formalin-fixed, paraffin-embedded biopsies using a widely available commercial antibody. A semi-quantitative analysis was made using a scoring system of 0, 1, 2, and 3, for increasing intensity of staining. High VEGF expression was associated with a poor prognosis. A univariate log rank analysis found a significant relationship with overall survival (P = 0.0008) and metastasis-free survival (P = 0.0062), but not local control (P = 0.23). There was no correlation between VEGF expression and disease stage, tumour differentiation, patient age, or tumour radiosensitivity (SF2). In a Cox multivariate analysis of survival VEGF expression was the most significant independent prognostic factor (P = 0.001). After allowing for VEGF only SF2 was a significant prognostic factor (P = 0.003). In conclusion, immunohistochemical analysis of VEGF expression is a highly significant and independent prognostic indicator of overall and metastasis-free survival for patients treated with radiotherapy for advanced carcinoma of the cervix. It is also a rapid and easy method that could be used in the clinical setting, to identify patients at high risk of failure with conventional radiotherapy who may benefit from novel approaches or chemoradiotherapy.