The effect of EGFR-related tyrosine kinase activity inhibition on the growth and invasion mechanisms of prostate carcinoma cell lines.

Increased urokinase plasminogen activator (uPA) levels and epidermal growth factor receptor (EGFR)-related tyrosine kinase activity are associated with poor prognosis in several cancers. We studied the effect of epidermal growth factor (EGF) and a specific inhibitor of EGFR, ZM252868, on the growth and invasiveness of the prostate cancer cell lines PC3 and DU145. PC3 cell growth was stimulated by exogenous EGF but DU145 cell growth was not. EGFR-specific tyrosine kinase inhibitor significantly inhibited the growth of both cell types. EGF increased uPA protein level and uPA activity in both cell types. EGF stimulation also resulted in increased uPAR transcript in both cell lines. uPA production and activity were suppressed by the inhibitor to well below the levels in control cells. Matrigel invasion of PC3 cells was increased by EGF. ZM252868 also reversed the EGF-stimulated matrigel invasion by PC3 cells. Our results indicate that EGF is a potent stimulative agent for both growth and invasion in prostate cancer cells, and that targeting the EGFR function inhibits not only tumor growth but also invasiveness.

Transforming growth factor beta1 stimulates urokinase plasminogen activator system on prostate cancer cells.

The effect of TGFbeta1 on the proliferation and plasminogen activator system (PA) of two prostate carcinoma cell lines, PC3 and DU145, was investigated. PA, particularly urokinase plasminogen activator (uPA), has been implicated in extracellular proteolysis, local invasiveness, metastatic spread and angiogenesis. High levels of uPA and plasminogen activator inhibitor-1 (PAI-1) correlate with poor prognosis in several cancers. TGFbeta1 had no significant effect on the proliferation of either cell line. TGFbeta1 increased the production of uPA in PC3 and DU145 cells. Despite the very low PAI-1 protein levels in both cell lines, TGFbeta1 treatment resulted in a remarkable increase in PAI-1 secretion. PAI-2 protein was also increased by 59% in the PC3 cells. A divergent effect of TGFbeta1 on the uPA enzyme activity was observed (28% decrease in PC3 and 131% increase in DU145 cells). Overall, TGFbeta1 treatment did not affect the invasion of reconstituted basement membrane of PC3 cells. In addition to the uPA:PAI-1 ratio, the presence of PAI-2 may be an important factor in the determination of metastatic sites for prostate cancer cells. In conclusion, the potential contribution of TGFbeta1 to tumor invasion may be considered as positive, based on both loss of growth inhibition and stimulation of components of the invasive system of prostate carcinoma.

Adjuvant ovarian ablation vs CMF chemotherapy in premenopausal breast cancer patients: trial update and impact of immunohistochemical assessment of ER status.

This trial, initiated in 1980, examined the relative values of adjuvant ovarian ablation and chemotherapy comprising cyclophosphamide, methotrexate and 5-fluorouracil (CMF) in premenopausal women with pathological stage II breast cancer. With median follow-up for patients still alive of 13.9 years, there is no difference in survival between women receiving ovarian ablation and CMF (hazard ratio 1.01; 95% CI: 0.74, 1.37). Tumour oestrogen receptor (ER) status was assessed at the time using biochemical ligand-binding assay and retrospectively by immunohistochemistry (IHC). Agreement between these two methods was only fair, but both confirmed the importance of ER status in determining appropriate adjuvant systemic therapy. A statistically significant interaction between IHC quick score and treatment (P=0.001) showed ovarian ablation was more beneficial for patients with a positive quick score, whereas women with a quick score of 0 had a significantly higher risk of death with ovarian ablation (2.33; 95% CI: 1.30, 4.20). We have shown that IHC identifies women with ER 'poor' tumours for whom endocrine manipulation is not appropriate.

What the clinician needs from the pathologist: evidence-based reporting in breast cancer.

Histopathology has a vital role in determining breast cancer management and pathologists must be part of the clinical team. Carcinoma size, grade, and especially lymph node status remain the best available prognostic factors. Metastatic carcinoma in axillary nodes is more important than any other prognostic factor presently available. ER status is an important predictor of response to endocrine manipulation, but its independent prognostic significance, and that of micrometastatic disease, circulating carcinoma cells and other molecular factors, even well-studied ones such as HER2 status, are less clear. Pathology is the first clinical speciality to subject its practice to rigorous scientific analysis, and it has stood up well. However, workers without appropriate experience in Pathology or scientific design have created difficulties by undertaking poorly planned studies with ill-defined end-points, lacking appropriate quality control. New analytical techniques and therapeutic targets make it essential that we learn from past mistakes and integrate pathologists into the research teams pursing clinical trials and the assessment of new bio-markers. Without this, input resource will be wasted on false leads that could have been curtailed. Morphology alone will not be enough to select patients likely to benefit in trials of new therapies, but selection 'tests' must be appropriate. The confusion of tests for selection of patients to receive Herceptin shows what happens when this process fails. Much of the microarray data being put into data-bases has no quality control, and meta-analysis of this data will produce even more conflict than the clinical trials. This can be avoided, as the ability to standardise is available.

Inhibition of transforming growth factor alpha (TGF-alpha)-mediated growth effects in ovarian cancer cell lines by a tyrosine kinase inhibitor ZM 252868.

The modulating effects of the epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor ZM 252868 on cell growth and signalling have been evaluated in four ovarian carcinoma cell lines PE01, PE04, SKOV-3 and PE01CDDP. Transforming growth factor alpha (TGF-alpha)-stimulated growth was completely inhibited by concentrations > or =0.3 microM in the PE01 and PE04 cell lines and by > or =0.1 microM in SKOV-3 cells. TGF-alpha inhibition of PE01CDDP growth was reversed by concentrations > or =0.1 microM ZM 252868. TGF-alpha-stimulated tyrosine phosphorylation of both the EGF receptor and c-erbB2 receptor in all four cell lines. The inhibitor ZM 252868, at concentrations > or =0.3 microM, completely inhibited TGF-alpha-stimulated tyrosine phosphorylation of the EGF receptor and reduced phosphorylation of the c-erbB2 protein. EGF-activated EGF receptor tyrosine kinase activity was completely inhibited by 3 microM ZM 252868 in PE01, SKOV-3 and PE01CDDP cells. These data indicate that the EGF receptor-targeted TK inhibitor ZM 252868 can inhibit growth of ovarian carcinoma cells in vitro consistent with inhibition of tyrosine phosphorylation at the EGF receptor.

Increased use of immunohistochemistry for oestrogen receptor measurement in mammary carcinoma: the need for quality assurance.

This paper outlines the changes which have occurred over the last 25 years in the methods employed for the measurement of oestrogen receptors to aid the management of women with breast cancer. Immunohistochemistry is now the method of choice and knowledge of oestrogen receptor status is being used with increasing frequency for the selection of adjuvant treatment as well as for the treatment of metastatic disease. It is essential that good quality assurance procedures are established so that results are reproducible and can be used with confidence in individual centres as well as being comparable with those produced elsewhere. A retrospective study of 170 women with metastatic breast cancer provides the basis for a discussion on the advantages and pitfalls of the immunohistochemical assay. Particular emphasis is paid to the choice of cut-off and how the results may be applied in patient management.

Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): report of an EORTC/BIOMED-1 workshop.

The urokinase-type plasminogen activator (uPA) is considered to play a key role in the process of invasion and metastasis. In several independent studies, in a variety of cancer types (e.g. of the breast, colon, stomach, lung, ovary), high antigen levels of uPA in tumour extracts have been associated with rapid disease progression. In these studies, different sets of antibodies and standards (often as commercially available uPA ELISA kits) have been used. The standards provided with the different uPA ELISA kits are different from each other in both composition and source. In addition, the different uPA ELISA kits use antibodies which differ in specificity and affinity for the various forms of uPA including pro-uPA, HMW-uPA, LMW-uPA, the aminoterminal fragment (ATF) and complexes with inhibitors (PAI-1 and PAI-2) and the receptor (uPAR). Further, the composition of tumour tissue extraction buffers differ significantly among the published studies. Thus, it is not surprising that the ranges of cytosolic uPA levels reported differ considerably even when measured within the same tumour type. These discrepancies led the EORTC Receptor and Biomarker Study Group, in conjunction with the BIOMED-1 consortium on 'Clinical Relevance of Proteases in Tumour Invasion and Metastasis', to organise a workshop to study the characteristics associated with six different uPA immunoassays (ELISA) used in clinical studies reported in the literature. Although the absolute uPA antigen values measured with the respective uPA ELISA kits differed, high correlations were obtained for any two of the four uPA ELISA kits finally applied to sets of breast cancer cytosol preparations. The preparations used at present as standards in the various uPA ELISA kits are not representative of actual human breast cancer cytosols. Thus absolute standardisation is only possible by using a common reference sample (breast cancer cytosol) and similarly composed ELISA uPA kits. Then it will be possible to generate comparable data on clinical tissue as well as to check for batch-to-batch variations within particular ELISA kits.

Detection and distribution of heat shock proteins 27 and 90 in human benign and malignant prostatic tissue.

OBJECTIVE: To determine whether it is possible to predict the behaviour of prostate tumours by identifying cellular characteristics, specifically specific heat shock proteins (HSPs). MATERIALS AND METHODS: An immunohistochemical study staining for HSP 27 and 90 was undertaken on 15 benign and 13 malignant samples of freshly frozen prostatic tissue obtained from patients with a similar age range in each group (benign, mean age 71.6 years, range 61-86; malignant, mean age 72.7 years, range 58-87). Gleason scores for the tumours ranged from 2 to 8. RESULTS: Consistent patterns of cytoplasmic staining were seen in all sections of tissue from benign prostatic hyperplasia (BPH). The stroma stained strongly positive for HSP 27, but negatively for HSP 90 and glandular epithelium showed positive apical staining for both HSPs. Stromal patterns in prostatic carcinoma tissue were similar to that of BPH tissue for both HSP 27 and 90. Areas of prostatic intra-epithelial neoplasia stained as strongly as did adjacent areas of BPH. For HSP 27, there was varied staining of individual epithelial cells, suggesting cellular heterogeneity, with an apparent reduction in staining with increasing Gleason score and invasiveness. For HSP 90, this pattern was less marked, with a predominance for positive staining throughout all grades of carcinoma. CONCLUSIONS: The distribution of HSPs, primarily HSP 27, may aid in identifying different cell populations within prostatic carcinomas and thus help forecast biological behaviour.

Steroid-growth factor interaction in human prostate cancer. 2. Effects of transforming growth factors on androgen metabolism of prostate cancer cells.

The ability of human prostate cancer cells to metabolize androgens was assessed through administration of physiological concentration (0.5-10 nM) of tritiated testosterone (T) as precursor and one-step analysis of both T degradation and products' formation by reverse-phase HPLC and on-line radioactive detection after either 24 h or 72 h incubation. Overall, different prostate cancer cells degraded T quite differently, favoring alternatively reductive or oxidative metabolic pathways. In particular, both LNCaP and DU145 cells retained high levels of unconverted T, with a limited production of androstenedione and its 17-keto derivatives and relatively high amounts of dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha-diol). In contrast, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione and 17-keto metabolites, while neither dihydrotestosterone nor 3 alpha-diol were detected after short or longer incubation times. The effects of both TGF alpha (50 ng/mL) and TGF beta 1 (5 ng/mL) on rates and direction of T metabolism were also explored. In LNCaP cells TGF alpha induced a significant (P < 0.04) decrease of the reductive metabolism of T with a corresponding enhancement of the oxidative pathway (P < 0.002), while TGF beta 1 did not significantly affect T metabolism. On the other hand, both reductive and oxidative pathways were only partially influenced by either growth factor in DU145 and PC3 cells, although TGF alpha significantly raised 5 alpha-androstanedione formation and reduced androsterone production in DU145 cells. All the above evidence was confirmed at both 24 h and 72 h or using increasing doses of TGF alpha and TGF beta 1, a peak activity of 50 ng/mL and 5 ng/mL, respectively, being generally encountered. Overall, our data suggest that TGFs may have a role in the growth regulation of hormone-responsive prostate tumor cells through changes of the intracellular contents of biologically active androgen metabolites.

Conclusions and recommendations from the Helene Harris Memorial Trust Fifth Biennial International Forum on Ovarian Cancer, May 4-7, 1995, Glasgow, UK.

The Fifth Biennial International Forum on Ovarian Cancer was held by the Helene Harris Memorial Trust in Glasgow, UK. The main points of the presentations given by the invited speakers, together with the fruits of extensive discussions are presented here as a series of conclusions and recommendations which should be given consideration by all those having an interest in researching or treating ovarian cancer. The individual points are grouped into topics considering whether there is an identifiable ovarian pro-cancer, whether ovarian cancer is preventable, advances in familial ovarian cancer, its molecular genetics, ways of optimizing treatment, obstacles to successful treatment and approaches to gene therapy.

Growth factors in human ovarian follicle fluid and growth factor receptors in granulosa-luteal cells.

The levels of oestradiol (E2), progesterone (P4), transforming growth factor alpha (TGF alpha), transforming growth factor beta 2 (TGF beta 2), insulin-like growth factor I (IGF-I), platelet-derived growth factor AB (PDGF-AB) and epidermal growth factor (EGF) were measured in follicular fluids obtained from patients undergoing ovarian stimulation as part of an in vitro fertilisation program. Each of the substances was detected in all of the fluid samples tested, except TGF alpha (which was detected in 90% of samples tested), PDGF-AB (70%) and EGF (2%). Comparisons were made between each of these factors, follicular maturity, successful oocyte recovery and the outcome of fertilisation and embryo transfer. No statistically significant correlations were found. The presence of receptors for EGF, IGF-I and PDGF in extracts from granulosa-luteal cells isolated from follicular fluids was detected by means of Western blotting. The co-localisation of these growth factors and their receptors within the ovarian follicle suggests a likely role in control of follicular development.

Cyclic sequential endocrine therapy for advanced breast cancer using a combination of tamoxifen and megestrol acetate.

A cyclical, sequential combination of tamoxifen and megestrol acetate (group B) was compared with conventional therapy (tamoxifen alone, group A) in 261 breast cancer patients. There was no statistically significant difference between groups for overall response rate (complete+partial response: group A, 35.8%, group B, 40.8%; p = 0.505) or for median response duration in responders (group A, 128 weeks, group B, 136 weeks; p = 0.488). Median survival from randomization was longer in those patients receiving sequential therapy (group A, 90 weeks, group B, 134 weeks) with a significantly lower relative death rate (group B/group A = 0.67; p = 0.011). This survival benefit appears to be due to a delay in progression among nonresponders in the sequential therapy group.

Steroid-growth factor interaction in human prostate cancer. 1. Short-term effects of transforming growth factors on growth of human prostate cancer cells.

In order to better define potential mechanisms of growth regulation in human prostate cancer cells, we have compared biological responses (such as short-term response to both transforming growth factor alpha and beta; TFG alpha and TFG beta) in relation to hormone sensitivity of LNCaP, DU145, and PC3 cells. Androgen receptor (AR) and epidermal growth factor receptor (EGF-R) content of each cell line was also investigated. In addition, expression of EGF, TGF alpha, and TGF beta was evaluated through immunofluorescent staining. Growth of androgen non-responsive PC3 cells was stimulated by TGF alpha (about 35%) and inhibited by TGF beta (more than 50%), with respect to controls, after 48 h exposure. Conversely, AR-positive, hormone-responsive LNCaP cells proved to be poorly sensitive, at least short-term, to either growth factor. Furthermore, high levels of both EGF-R and TGF alpha, and a fairly high amount of EGF, were found in DU145 cells and, to a lesser extent, in LNCaP cells; in contrast, PC3 cells exhibited low expression levels of both receptors (EGF-R) and ligands (EGF, TGF alpha), but displayed remarkable TGF beta binding and relatively high levels of endogenous TGF beta. Overall, these results suggest a differential sensitivity to TGF alpha and TGF beta by prostate cancer cells; TGF alpha response seems not to be proportional to the EGF-R content of individual cell lines.

Immunoreactivity of antibodies to epidermal growth factor, transforming growth factors alpha and beta, and epidermal growth factor receptor in the premenopausal ovary.

This study provides a valuable insight into the localization of growth factors in paraffin sections of human ovarian tissue. Antibodies to epidermal growth factor (EGF), transforming growth factors alpha and beta (TGF alpha and beta) and epidermal growth factor receptor (EGFR) were applied to paraffin sections of 16 cases of formalin-fixed normal or benignly abnormal ovarian tissue. All growth factor antibodies reacted with theca, but not granulosa cells, whilst the antibody to EGFR reacted with both types of follicular cells and was weakly reactive in ovarian stroma. There were no discernible qualitative changes in reactivity during the follicular cycle. These immunohistochemical findings generally support previously published molecular and biochemical data from tissue culture. One exception is in the observation of immunoreactivity to EGF in theca and granulosa cells. This may be due to differences in sensitivity of the methods in use. The possibility of a cross-reaction of the anti-EGF antibody with TGF alpha is also discussed. This study provides evidence for both paracrine and autocrine roles for growth factors in folliculogenesis.

Growth factor content in normal and benign ovarian tumours.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) content was measured in normal ovaries and benign ovarian tumours. Epidermal growth factor was present in 12.7% of normal ovaries, with a range 0.030-0.533 ng/mg DNA, and in 31.8% of benign ovarian tumours, with a range 0.1335-2.080 ng/ml DNA. TGF alpha was present in 84.5% of normal ovaries, with a range of values from 0.037-18.2 ng/mg DNA, and in 84.1% of benign ovarian tumours, with a range of 0.083-195 ng/mg DNA. The TGF alpha content in post menopausal benign ovarian tumours was significantly higher (P < 0.0001) than TGF alpha in the pre-menopausal normal ovarian group. The frequency of detection and levels of TGF alpha measured were significantly higher than those of EGF in the normal ovary group (P < 0.001) and also in the benign ovarian group (P < 0.005). We conclude that TGF alpha is the predominant growth factor present in normal ovaries and benign ovarian tumours.

Epidermal growth factor receptor in normal ovaries and benign ovarian tumours.

Epidermal growth factor receptor (EGFR) was assayed in 52 women who had normal ovaries removed at hysterectomy and in 30 women with benign ovarian tumours. The histology of each ovary was recorded. A single point screen was performed on all samples and in positive cases a full Scatchard analysis. EGFR was present in 8 of 52 normal ovaries (15.4%) and 3 contained the high-affinity component while 5 had the low affinity component. In the benign ovarian tumour group 4 of 30 tumours (13.3%) had receptor present, one was high affinity and 3 were low affinity in type. We can conclude that EGFR is detectable only at low frequency in normal and benign ovarian tumours.

Local hyperthermia for prostatic disease: in vitro studies on human prostatic cancer cell lines.

Local hyperthermia for benign and malignant prostatic disease remains largely empirical. In an attempt to understand the biological action of hyperthermia, and its potentiation by antiandrogen seen in clinical practice, the interaction of the two has been studied in prostatic cancer cell lines. Human prostatic cancer cell lines LNCaP and DU 145 were studied to examine the effects of heat shock treatment (HST), androgen (5 alpha-dihydrotestosterone: 5 alpha DHT) and antiandrogen (hydroxyflutamide: OH-Flut) on cell growth and survival. Response (measured as increased DNA content) to 5 alpha DHT demonstrated that LNCaP was androgen sensitive, whereas DU 145 was androgen insensitive; OH-Flut stimulated LNCaP growth but had no effect on DU 145 growth. Thermotolerance was exhibited by DU 145 cells but not by LNCaP cells. The combination of HST followed by OH-Flut markedly reduced survival of LNCaP cells compared with HST alone. This effect was not observed in DU 145 cells. The enhanced cytotoxic effect of antiandrogen and hyperthermia could minimise the effect of thermotolerance in malignant cells surviving initial hyperthermia treatment and might suggest real clinical value for the combination or sequence.

Ki-67 antibody immunostaining in benign and malignant human prostatic disease.

Indices of mitotic potential may improve prognostic discrimination in patients with malignant disease. Ki-67 is a monoclonal antibody directed against an unknown proliferation antigen which has been shown to be a measure of mitotic potential. Sixty-four benign and eighty malignant prostatic biopsies were stained with the Ki-67 antibody. Nuclear and cytoplasmic staining was identified in benign and malignant biopsies using immunoalkaline phosphatase and immunoperoxidase staining reactions. Nuclear staining was identified in 14 benign and 44 malignant biopsies. Nuclear staining for Ki-67 was seen in 36% of biopsies with Gleason histological score (GHS) 2-4, 71% with GHS 5-7 and 62% with GHS 8-10. Nuclear staining was associated with advanced local disease stage, but not with metastatic disease stage. Clinical follow-up is required to establish the value of Ki-67 immunostaining as a prognostic determinant in prostatic cancer.

A comparison of biochemical and immunohistochemical assessment of EGFR expression in ovarian cancer.

The presence of epidermal growth factor receptor (EGFR) was determined both by immunohistochemistry and ligand binding assay in 118 samples from 96 cases of ovarian cancer. EGFR was present in 47.5% of tumours biochemically and in 39.8% of tumours analysed immunohistochemically. The concordance rate for the techniques varied between 40% in endometrioid carcinomas to 85.7% in undifferentiated carcinomas with an overall concordance of 69.5% (p < 0.001). There was no significant difference between the presence of the high, low or high plus low affinity receptor components and tumour immunoreactivity. Although the ligand binding assay is more sensitive than immunohistochemistry for detecting the epidermal growth factor receptor, some cases are positive only on immunohistochemical screening. We would recommend that both techniques should be performed in prospective studies in order to elucidate the role of EGFR expression in ovarian cancer.