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.

High tumor angiogenesis is associated with poorer survival in carcinoma of the cervix treated with radiotherapy.

The purpose of this study was to examine the relationship between tumor angiogenesis and prognosis in carcinoma of the cervix treated with radiotherapy with a median follow-up time of 55 months. A retrospective study was carried out on 111 patients. Formalin-fixed, paraffin-embedded tumor biopsies were stained with anti-factor VIII using immunohistochemistry. Tumor angiogenesis was assessed by scoring the distance to the closest microvessel from random points within the tumor and the intratumor microvessel density (IMD) in the areas of highest neovascularization. High vascularity, as measured by both methods, was associated with a poor prognosis but was only significant for IMD. The 5-year survival rates for tumors with high versus low values were 50 and 65%, respectively. IMD was a significant prognostic factor within a Cox multivariate analysis. Higher tumor vascularity was associated with lower overall survival and locoregional control, but this association was not significant in the case of metastasis-free survival. The method used to assess tumor vascularity is important. The level of angiogenesis in carcinoma of the cervix is an independent prognostic parameter.

A functional interaction between the mesolimbic dopamine system and vasopressin release in the regulation of blood pressure in conscious rats.

The aim of the present study was to further characterize the involvement of the mesolimbic dopamine system in central blood pressure regulation, with particular emphasis on the interaction of this system with the effects of circulating vasopressin. In conscious rats we stimulated the release of endogenous dopamine from mesolimbic/mesocortical terminals by administration of the substance P analogue DiMe-C7 ([pGlu5, MePhe8, Sar9]-Substance P5-11; 10 nmol) into the ventral tegmental area. Chemical stimulation of the ventral tegmental area resulted in a significant increase in blood pressure and heart rate. These effects were prevented by either bilateral electrolytic lesions of the hypothalamic supraoptic nucleus or by systemic pretreatment with the dopamine D2 receptor antagonist raclopride (0.5 mg/kg). Stimulation of the ventral tegmental area also produced a marked increase in the expression of the proto-oncogene c-fos in the supraoptic nucleus and a significant increase in plasma vasopressin levels, suggesting activation of vasopressinergic neurons in this nucleus. However, this effect of stimulation of the ventral tegmental area was not significantly inhibited by pretreatment with raclopride. We suggest that the effects on blood pressure and heart rate of stimulation of the ventral midbrain by micro-injection of DiMe-C7 are the result of combined activation of both dopaminergic and non-dopaminergic cell bodies in this region. Stimulation of non-dopaminergic cells in the ventral midbrain may induce a moderate increase in plasma vasopressin levels by activation of the supraoptic nucleus. An additional stimulation of dopaminergic cells in the ventral midbrain allows the increase in circulating vasopressin levels to become manifest as a pressor response, possibly by inhibition of vasopressin-induced facilitation of baroreflex responses.

Apoptosis, intrinsic radiosensitivity and prediction of radiotherapy response in cervical carcinoma.

Apoptosis is an important mechanism of cell death in tumours and it is seen both prior to and following radiotherapy. In this study patients with proven carcinoma of the cervix had measurement made of the percentage of apoptotic cells (apoptotic index or AI) in pre-therapy biopsies. Measurements of intrinsic radiosensitivity (SF2), already shown to be a predictor of outcome, had previously been made on the same pre-therapy biopsies. Mitotic index (MI) and Ki-67 antigen staining were also recorded as markers for proliferation. Patients were divided into those with an AI above or below the median and in general increasing apoptosis was associated with poor prognosis. The 5-year survival rate for tumours with an AI below the median was 79% and was significantly greater than the rate of 47% for those with an AI above the median (p = 0.003). There was also a significantly increased 5-year local recurrence-free rate for patients with an AI below the median compared with those with an AI above the median (79 versus 61%, p = 0.012). In addition, AI and SF2 acted as independent prognostic indicators. Patients with both an SF2 and AI value above the median did badly (25% 5-year survival, 46% local control) compared with those with an SF2 and AI below the median (80% 5-year survival, 100% local control). Apoptosis showed correlation with MI (n = 66, r = 0.34, p = 0.002) and cell staining for the Ki-67 antigen (n = 57, r = 0.25, p = 0.03), but neither MI nor Ki-67 were related to patient outcome. This suggests that while apoptosis may be a reflection of tumour proliferation this cannot in itself explain the ability of apoptosis to predict clinical outcome for this series of patients. The study raises the possibility of AI and SF2 being used together as predictors of tumour response to radiotherapy.

Prognostic significance of c-erbB-2 protein expression in carcinoma of the cervix treated with radiotherapy.

PURPOSE: The relationship was studied between c-erbB-2 expression and outcome in 107 carcinomas of the cervix treated with radical radiotherapy. METHODS: Formalin-fixed, paraffin-embedded sections were stained by immunohistochemistry for over-expression of the c-erbB-2 protein. A retrospective study of treatment outcome was made on patients with a median follow-up of 55 months. RESULTS: Patients with c-erbB-2-positive tumours had a significantly worse overall survival rate than those with c-crbB-2-negative tumours (P=0.019). Metastasis-free survival (i.e. recurrence outside the radiotherapy field) was also significantly worse (P < 0.001) but there were no differences in local control (i.e. recurrence within the radiotherapy field) rates (P=0.24). Bivariate log-rank analyses showed that the prognostic value of c-erbB-2 expression for metastasis-free survival was independent of disease stage, histological grade, patient age, tumour size and tumour radiosensitivity. A combination of two biological parameters yielded a high discrimination between outcome groups. Women with radiosensitive and c-erbB-2-negative tumours had a 5-year metastasis-free survival level of 70% compared to 33% for women with radioresistant, c-erbB-2-positive tumours. CONCLUSIONS: c-erbB-2 expression is an important prognostic factor for determining tumour recurrence outside the treatment field in cervix carcinomas treated with radiotherapy.

Assessment of light scatter by nucleoids as a rapid predictive assay of radiosensitivity.

A study has been made of the practicality of using the assay of light scatter by nucleoids as a rapid predictive test of cellular radiosensitivity. With this technique the effect of irradiation on DNA organization is measured using flow cytometry after staining irradiated nucleoids with a high concentration of ethidium bromide. Damaged nucleoids fail to respond to the ethidium bromide-induced contraction and scatter more forward-angle light than less damaged nucleoids. Seventeen different cell lines were assessed using a single lysis condition and radiation dose. Significant differences in the levels of radiation-induced forward-angle light scatter by nucleoids were seen between CHO cells and cells of two radiosensitive mutant cell lines (xrs-6, EM9), and between cells of two ovarian carcinoma lines that showed marked differences in radiosensitivity measured using a clonogenic assay. However, other cell lines which differed in clonogenic radiosensitivity showed similar forward-angle light scatter by nucleoids. When all 17 cell lines were included in the analysis, there was no correlation between measurements of radiosensitivity by assays of clonogenicity and light scatter by nucleoids. In addition, although intraexperimental variation was small, the level of interexperimental variability was only slightly smaller (coefficient of variation of 13%) than the degree of heterogeneity observed between the different cell lines (coefficient of variation of 16%). These findings support the notion for a role of nuclear structure as a determinant of intrinsic radiosensitivity for some cell lines but suggest that for others there must be additional, more dominant factors.

The intrinsic radiosensitivity of normal and tumour cells.

PURPOSE: To examine whether in vitro measurements of normal and tumour cell radiosensitivity can be used as prognostic factors in clinical oncology. MATERIALS AND METHODS: Stage I-III cervix carcinoma patients were treated with radical radiotherapy with a minimum of 3 years' follow-up. Lymphocyte and tumour radiosensitivities were assayed using, respectively, a limiting dilution and soft agar clonogenic assay to obtain surviving fraction at 2 Gy (SF2). The results were related, in an actuarial analysis, to late morbidity assessed using the Franco Italian glossary. RESULTS: Patients with radiosensitive lymphocytes had a significantly increased risk of developing late complications (n = 93, p = 0.002). Increasing tumour radiosensitivity was associated with an increased risk of morbidity (n= 113, p=0.032). A significant correlation was found between fibroblast and tumour cell radiosensitivity (r=0.57, p=0.03), but a weak inverse association was found between lymphocyte and tumour cell radiosensitivity (r= -0.32, p=0.03). Patients with radiosensitive lymphocytes and tumour cells had higher levels of late complications than those whose cells were radioresistant. CONCLUSION: The work described highlights the importance of cellular radiosensitivity as a parameter determining the clinical response to radiotherapy.

A serum-free medium for the Courtenay-Mills soft agar assay.

A serum-free medium has been developed to support the clonogenic growth in soft agar of human cervical carcinoma cell lines xenografted into nude mice. Using the Courtenay-Mills assay, colony-forming efficiencies and colony sizes equivalent to those obtained using Ham's F12 plus 15% fetal calf serum can be obtained. Validation of the assay using the developed medium was obtained through establishing linearity between the number of cells plated and colonies formed, and by producing radiation survival curves.

Inhibition of cardiac baroreflex sensitivity after central dopaminergic stimulation.

1. Stimulation of the mesolimbic dopamine system, by micro-injection of the substance P analogue [pGlu5,MePhe8,Sar9] substance P (DiMe-C7) into the ventral tegmental area induced a prolonged increase in blood pressure and circulating levels of vasopressin. 2. In the present study, this treatment produced a significant decrease of cardiac baroreflex sensitivity in conscious rats. After pretreatment with the dopamine receptor antagonist raclopride, central stimulation failed to produce any changes in baroreflex parameters. 3. The central dopamine-mediated decrease in baroreflex sensitivity may be involved in functionally potentiating the circulatory actions of vasopressin.

Immunocytochemical and lectin histochemical studies of plaques and tangles in Down's syndrome patients at different ages.

The presence of numerous senile plaques (SP) and neurofibrillary tangles (NFT) within association areas of the neocortex and within the hippocampus and amygdala, is generally regarded as providing the histopathological hallmarks of Alzheimer's disease (AD) (Mann, 1985). Similar pathological changes, to those of AD, are seen in the brains of nearly all persons with Down's syndrome (DS) who live beyond 40 years of age, though such features are only rarely seen before 20 years of age (Mann, 1988). Because of this similarity between AD and DS at middle age, DS has been considered (Mann, 1988) to provide a useful model for the pathological process of AD. Hence a study of DS patients at different ages (and in all of whom the pathological changes typical of AD would have been expected had they lived long enough) can provide important data concerning the time course of the acquisition and the morphological and biochemical genesis of SP and NFT in DS, with obvious implications as to the changes of AD itself. In this study, the brains of 24 patients dying, between the ages of 13 and 65 years, with DS have been examined for the presence and the morphological appearance of SP and NFT using various markers for structural or biochemical components known, from previous work on AD (Ihara, 1988; Ihara et al., 1986; Davies et al., 1988; Mann et al., 1988) to be associated with SP and NFT.

Effects of 4-hydroxytamoxifen and a novel pure antioestrogen (ICI 182780) on the clonogenic growth of human breast cancer cells in vitro.

We have investigated the effects on breast cancer cell growth of 4-hydroxytamoxifen (4OHT), a conventional antioestrogen with agonist activity, and 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulphinyl)nonyl]oestra- 1,3,5,(10)- triene-3,17 beta-diol (ICI 182780), a novel, pure antioestrogen, using established human breast cancer cell lines and cancer cells obtained directly from breast cancer patients with malignant pleural effusions who had relapsed on tamoxifen. The effects of the two agents were assessed using the Courtenay-Mills clonogenic assay, which measures the growth of single cancer cells as colonies suspended in soft agar. The standard assay was modified by the use of defined serum- and phenol red-free growth medium. The growth of oestrogen receptor (ER)-positive MCF-7 cells in the assay was oestrogen responsive. Both antioestrogens inhibited the stimulatory effects of 1 nM oestradiol, but ICI 182780 caused significantly greater inhibition than 4OHT at 0.1-1.0 microM concentrations. In the absence of oestradiol, 4OHT but not ICI 182780 caused significant stimulation of colony formation at low (0.01-1.00 nM) concentrations. Neither antioestrogen had any effects on colony formation by the ER-negative Hs578T cell line. Successful colony formation was obtained in primary cultures from six out of eight malignant effusions. Colony formation was significantly stimulated by 0.1 nM oestradiol in four cases and by 10 nM 40HT in two cases. In contrast, ICI 182780 exhibited no intrinsic stimulatory activity and significantly inhibited both oestradiol- and 4OHT-stimulated cell growth. We conclude that the agonist activity of 4OHT and other conventional antioestrogens may cause treatment failure in some patients by stimulating breast cancer cell growth. The new, pure antioestrogen ICI 182780 is a more potent oestrogen antagonist than 4OHT and exhibits no growth-stimulatory activity. This agent may therefore offer therapeutic advantages over conventional antioestrogens in patients with advanced breast cancer and may be effective after conventional agents have failed.

Tumour vascularity is a significant prognostic factor for cervix carcinoma treated with radiotherapy: independence from tumour radiosensitivity.

The aim of the study was to investigate the relationship between intrinsic radiosensitivity and vascularity in carcinoma of the cervix given radiotherapy, and assess whether more refined prognostic information can be gained by combining the two parameters. A retrospective study was carried out on 74 patients with locally advanced carcinoma of the cervix. Formalin-fixed, paraffin-embedded tumour biopsies were stained with anti-factor VIII using immunohistochemistry. Vascularity was scored using the intra-tumour microvessel density (IMD), or 'hot-spot', technique. For the same patients, the measurement of intrinsic radiosensitivity (SF2) had been made previously on the same pretherapy samples. Patients were stratified by the median IMD and SF2 scores. Women with radioresistant and highly vascular tumours had poorer 5-year survival (P = 0.0005, P = 0.035 respectively) and local control (P = 0.012, P = 0.077 respectively) than those with radiosensitive and poorly vascular tumours. No significant correlation was seen between IMD and SF2. Multivariate analysis (including tumour stage and patient age) showed that only SF2 and IMD were significant prognostic factors for survival. Patients with both a radioresistant and highly vascular tumour had a 5-year survival level of 18% compared to 77% for those patients with a radiosensitive and poorly vascularized tumour. Tumour angiogenesis and cellular radiosensitivity are independent prognostic factors for cervix carcinoma treated with radiotherapy. Allowing for tumour radiosensitivity increases the prognostic significance of vascularity measurements in cervix tumours.

Levels of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (APE1, APEX, Ref-1) are associated with the intrinsic radiosensitivity of cervical cancers.

A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra- and intertumour heterogeneity were 62% and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r = 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The results suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.