Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor beta1.

We have previously demonstrated that peritumoral stromal matrix derived from prostate cancer patients who relapse after radical surgery contains elevated levels of versican. The purpose of this study was to determine whether prostate cancer cells control stromal cell secretion of versican. Serum-free conditioned medium from three prostate adenocarcinoma cell lines, LNCaP, PC3, and DU145, was added to cultures of fibroblasts established from prostatic tissue of patients with benign prostatic hyperplasia, and the medium was harvested at 24, 48, and 72 h. Immunoblotting with an antiversican core protein antibody revealed that prostatic fibroblast medium harvested at 72 h contained increased levels of versican after treatment with either LNCaP-, PC3- or DU145-conditioned medium (2.5-, 4.5-, and 5-fold, respectively) compared with control cultures. This increase in versican in the culture medium was not observed after coincubation with transforming growth factor beta1-neutralizing antisera. The results of this study suggest that prostate tumor cells induce host stromal cells to secrete increased versican levels via a paracrine mechanism mediated by transforming growth factor beta1.

Detection of discrete androgen receptor epitopes in prostate cancer by immunostaining: measurement by color video image analysis.

To determine whether multiple features of immunohistochemical staining of the androgen receptor (AR) in prostate cancer could reliably predict androgen dependence, tumor biopsy specimens from 30 patients (stages A-D2) were stained using anti-peptide antibodies to the amino- and carboxyl-terminal of the AR. Measurements were made of the mean area and total amount (i.e., integrated optical density) of AR staining in at least 20 fields per section using a color video image analysis system, and the mean intensity of AR staining per cell and the percentage of AR positive tumor cells were derived. Video image analysis measurement identified quantitative differences in AR staining between the two antibodies, suggesting that this approach may provide a means of identifying receptor variants in prostate tumors. The AR staining measurements were analyzed by discriminant function analysis to assign individual cases to good and poor clinical outcome groups. AR staining features measured with a single antibody (e.g., amino-terminal) were sufficient to predict outcome following hormonal therapy in stage D2 patients (predictive value, 1.0), whereas all features of AR staining measured with both antibodies were required for the entire patient group (predictive value, 0.97). The principal discriminant in both patient groups contributing to the correct assignment of outcome was the mean intensity of AR staining per cell. These findings suggest that AR staining features measured by video image analysis have the potential to predict outcome in prostate cancer.

Elevated levels of peritumoral chondroitin sulfate are predictive of poor prognosis in patients treated by radical prostatectomy for early-stage prostate cancer.

The disease course of localized prostate cancer is highly variable, and patients potentially curable by aggressive management are not readily identified by current clinical practice. Chondroitin sulfate (CS) glycosaminoglycan is a candidate biomarker as elevated levels of CS in peritumoral stroma of prostate cancer have been associated with prostate-specific antigen (PSA) failure. Immunoreactive CS was measured using image analysis of archived radical prostatectomy tissues, obtained from 157 men with a median of 47 months (range, 16-111 months) clinical follow-up. CS level, Gleason score, and preoperative serum PSA levels were independent predictors of PSA failure by Cox's multivariate analysis. Patients with low CS levels had significantly fewer PSA failures after radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot; 32% PSA failures at 5 years for CS mean integrated absorbance cut point < 7.0 versus 50% for CS > or = 7.0, P = 0.0001). In the subgroup of patients with preoperative serum PSA levels < 10 ng/ml, CS was particularly useful in discriminating retrospectively those patients most suited for surgery (Kaplan-Meier plot; 14% PSA failures at 5 years for CS mean integrated absorbance cut point < 7.0 versus 47% for CS > or = 7.0, P = 0.0001). We conclude that measurements of CS level can assist in predicting patient outcome after surgery. Additionally, our data suggest that the combination of CS and PSA measurements may improve outcome prediction for patients with intermediate Gleason scores.

EMS1 amplification can occur independently of CCND1 or INT-2 amplification at 11q13 and may identify different phenotypes in primary breast cancer.

Chromosome 11q13 is amplified in about 13% of primary breast cancers. CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding a filamentous actin binding protein, are favoured candidate onocogenes, whereas INT-2 is an unexpressed gene at this locus. In this study we tested the possibility that different regions of this large amplicon could be independently amplified and subsequently defined the phenotype of EMS1 amplified tumours in a series of 961 primary breast carcinomas. Using DNA slot blots, EMS1 was amplified in 15.2% of samples: 5.4% were coamplified for CCND1; 7.9% coamplified for INT-2 and 6.7% showed EMS1 amplification alone. The degree of amplification of CCND1 and INT-2 was highly correlated (P =0.0001). In contrast, no such relationship existed between EMS1 and CCND1 or INT-2 amplification, demonstrating independent amplification of EMS1 in 44% of amplified tumours. EMS1 amplification (> or = twofold increase in copy number) was positively correlated with patient age > or = 50 years (P = 0.025), ER positivity (P = 0.022), PgR positivity (P = 0.018), and was negatively correlated with HER-2/neu (c-erbB2) amplification (P = 0.01). In common with CCND1/INT-2, EMS1 amplification was associated with increased risk of relapse in patients with lymph node-negative disease (P = 0.028). In contrast, EMS1 and CCND1/INT-2 amplification appeared to confer different phenotypes in ER positive and negative tumours. A > or = threefold increase in EMS1 copy number was associated with an apparent increased risk of relapse and death in patients with ER negative tumours, but was without effect in ER positive tumours. In contrast, CCND1/INT-2 amplification had no effect in the patients with ER negative tumours but was associated with early relapse in ER positive patients. Thus EMS1 amplification may identify subgroups of breast cancer patients with increased probability of relapse and death distinct from those identified by CCND1/INT-2 amplification. Further studies are required to more clearly determine the functional consequences of EMS1 overexpression and a biological basis for the relationship between EMS1 amplification and phenotype in breast cancer.

Medroxyprogesterone acetate therapy in advanced breast cancer: the predictive value of androgen receptor expression.

PURPOSE: To determine the predictive value of androgen receptor (AR) levels in primary tumors of women who undergo medroxyprogesterone acetate (MPA) therapy for advanced breast cancer after relapse on tamoxifen adjuvant therapy. METHODS: Between 1984 and 1987 at Flinders Medical Centre, South Australia, 136 postmenopausal women received adjuvant tamoxifen therapy for lymph node-positive breast cancer. Estrogen receptor (ER), progesterone receptor (PgR), and AR levels, tumor size, and degree of axillary node involvement were determined at the time of diagnosis. The median follow-up period was 81 months; 89 women developed metastatic disease, 83 of whom subsequently received MPA (500 mg/d). The objective response rate ([RR] ie, complete response [CR] and partial response [PR]) and progression-free interval (PFI) were assessed in response to MPA therapy. Associations between RR, PFI, and primary tumor characteristics including ER, PgR, and AR levels were examined using the Mann-Whitney U test, Kaplan-Meier product-limit estimator, and Cox proportional hazards regression, as appropriate. RESULTS: Thirty-two of 83 patients (38.6%) responded to MPA. RR was significantly associated with the presence of AR (P < .001), but not with other primary tumor characteristics or duration of tamoxifen therapy. After initiation of MPA treatment, PFI increased with increasing concentration of AR in the primary tumor. CONCLUSION: Response to MPA after adjuvant tamoxifen treatment for lymph node-positive breast cancer was positively associated with AR level in the primary tumor. This finding suggests that MPA action in breast cancer may be mediated in part by the AR.

Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group.

PURPOSE: To determine prospectively the prognostic significance of HER-2/neu oncogene amplification in the primary tumors of breast cancer patients. METHODS: HER-2/neu amplification in tumor DNA was determined by the slot-blot technique in 1,056 patients with breast cancer (stage I to III) diagnosed between 1987 and 1990. Parameters such as estrogen receptor (ER) and progesterone receptor (PgR) levels, tumor size, axillary nodal involvement, tumor grade, and time to relapse were prospectively obtained. RESULTS: HER-2/neu oncogene amplification, > or = 2, > or = 3, and > or = 5 copy number, was detected in 21%, 11%, and 7% of patients, respectively. In a test set of 529 patients, Cox multivariate analysis showed HER-2/neu copy number > or = 3 or > or = 5 was associated with shorter disease-free survival (DFS) duration. HER-2/neu copy number > or = 3 correlated significantly with pathologic stage of disease, number of axillary nodes with tumor, histologic type, and absence of ER and PgR. For all patients, after a median follow-up duration of 39 months, Kaplan-Meier univariate analysis indicated that tumor oncogene copy number > or = 3 correlated with shorter DFS in both node-negative and node-positive patients. In Cox multivariate analysis, HER-2/neu copy number > or = 3 was associated with shorter DFS, independent of nodal status, ER level, and tumor size. CONCLUSION: Although the follow-up duration of this study is relatively short, we conclude that HER-2/neu amplification is an independent predictor of shorter DFS in both node-negative and node-positive patients.

Cyclin DI amplification is not associated with reduced overall survival in primary breast cancer but may predict early relapse in patients with features of good prognosis.

Amplification of chromosome 11q13 is frequently observed in human malignancies, including breast cancers. A candidate oncogene at this locus is the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. Because published data on the relationship between 11q13 amplification and prognosis in breast cancer have been controversial, we investigated the clinical significance of CCND1 amplification and its association with established clinicopathological features of prognosis in 1014 primary breast cancer patients. Amplification of the CCND1 gene and the INT-2/FGF-3 gene, which also maps to 11q13, was 10% and 17%, respectively. There were no associations between CCND1 or INT-2 amplification and patient age, tumor size, tumor grade, axillary lymph node status, HER/neu amplification, MIB-1 monoclonal antibody to Ki67 antigen count, or p53 expression. CCND1 amplification was predominantly observed in hormone receptor-positive tumors; at a copy number >/=3, CCND1 amplification was significantly correlated with both estrogen receptor (ER; P = 0.036) and progesterone receptor (P = 0.012) positivity. After a median follow-up period of 66 months, CCND1 or INT-2 amplification was not associated with significant increases in relapse or death from breast cancer. However, in the node-negative and ER-positive subgroups, there was a trend for an increased relapse rate in patients with INT-2 or CCND1 amplification. Thus, in this study, assessment of CCND1 or INT-2 amplification at 11q13 by slot-blot hybridization was of little use in determining phenotype or disease outcome in the whole group of patients but had a potential role in identifying a subset of poor-prognosis patients within the node-negative or ER-positive, good-prognosis groups. Because the prevalence of CCND1 amplification is much lower than the reported prevalence of cyclin D1 overexpression, additional studies are required to determine the true prognostic significance of altered cyclin D1 expression in breast cancer.

Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer.

Patients with clinically localized prostate cancer who might be cured by aggressive management are not easily identified using current clinical information. Additional, more accurate, biomarkers of tumor behavior need to be identified to improve clinical outcome. Our previous studies indicated that the concentration of the glycosaminoglycan chondroitin sulfate in prostatic stroma might be a useful biomarker of disease progression in early-stage prostate cancer. In this study, two chondroitin sulfate proteoglycans, versican and decorin, were investigated. Versican and decorin were immunolocalized to the periacinar and peritumoral fibromuscular stroma in sections of nonmalignant and malignant human prostate tissues. Video image measurements indicated that the concentrations of both proteoglycans were increased in the prostatic tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P = 0.0006). Cox's univariate analysis indicated that increases in versican concentration but not in that of decorin were associated with increased risk of prostate-specific antigen (PSA) progression. Versican concentration was compared with other clinical or biological features of prognosis in two-variable regression analyses. Versican and serum PSA concentrations were independent predictors of PSA progression. Versican was a stronger prognostic factor than tumor grade, and it could predict outcome for patients with moderately differentiated tumors. Patients with low versican concentration had significantly better progression-free survival than patients with high levels of versican (Kaplan-Meier plot, 89% versus 27% PSA progression-free at 5 years, respectively; P = 0.0001). We conclude that the measurement of prostatic concentrations of versican, a molecule with reported anticellular adhesive properties, may be a useful marker of disease progression in patients with early-stage prostate cancer and that further study of versican in other patient cohorts is warranted.

Elevated stromal chondroitin sulfate glycosaminoglycan predicts progression in early-stage prostate cancer.

Curative therapies for clinically localized prostate cancer have significant morbidity, and those patients who might be cured by aggressive management are not easily identified using current clinical information. Better biomarkers of tumor behavior need to be identified to improve clinical outcome. Chondroitin sulfate (CS), a glycosaminoglycan, may be a potentially useful biomarker as it is known to influence cell growth and differentiation and might influence malignant progression. In this study, CS was immuno-localized to the periacinar and peritumoral fibromuscular stromal tissue of nonmalignant and malignant prostates. The CS concentration was increased in the prostate tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P < 0.0001). Using Cox's univariate analysis, CS concentration, tumor grade, preoperative serum prostate-specific antigen (PSA), extracapsular extension of disease, positive surgical margins, and patient age were associated with an increased risk of PSA failure. The CS concentration was compared with the other features in two-variable regression analyses. CS and preoperative serum PSA concentrations were independent predictors of PSA failure. CS was a stronger prognostic feature than tumor grade and could predict outcome for patients with moderately differentiated tumors. Patients with a low CS concentration had significantly better progression-free survival following radical prostatectomy than patients with high levels of CS (Kaplan-Meier plot, 91% versus 49% PSA progression free at 5 years, respectively, P = 0.0038). Only postoperative pathological indices (extracapsular extension, surgical margins) were stronger predictors than CS. We conclude that measurement of prostatic CS concentrations at diagnosis may allow stratification of patients with early-stage prostate cancer for adjunctive or alternate therapies.

Mutations at the boundary of the hinge and ligand binding domain of the androgen receptor confer increased transactivation function.

The androgen receptor (AR), a member of the steroid receptor superfamily of nuclear transcription factors, mediates androgen signaling in diverse target tissues. Here we report AR gene mutations identified in human prostate cancer and the autochthonous transgenic adenocarcinoma of the mouse prostate model that colocate to residues (668)QPIF(671) at the boundary of the hinge and ligand-binding domain, resulting in receptors that exhibit 2- to 4-fold increased activity compared with wild-type AR in response to dihydrotestosterone, estradiol, progesterone, adrenal androgens, and the AR antagonist, hydroxyflutamide, without an apparent effect on receptor levels, ligand binding kinetics, or DNA binding. The expression of these or similar variants could explain the emergence of hormone refractory disease in a subset of patients. Homology modeling indicates that amino acid residues (668)QPIF(671) form a ridge bordering a potential protein-protein interaction surface. The naturally occurring AR gene mutations reported in this study result in decreased hydrophobicity of this surface, suggesting that altered receptor-protein interaction mediates the precocious activity of the AR variants.

Effects of ageing and hormonal manipulations on the level of oestrogen receptors in the guinea-pig prostate.

Cytosolic oestrogen receptor levels in guinea-pig prostate tissue were found to decrease with increasing age, irrespective of whether the binding was expressed relative to cytosolic protein or cellular DNA. This decrease in oestrogen receptor levels was also observed using enriched fibromuscular stromal tissue prepared by mechanical fractionation of the prostate. The most pronounced change in cytosolic oestrogen receptor levels (from 133 to 35 fmol/mg protein) occurred at the onset of puberty. The pubertal decrease in receptor levels could not be attributed to an increase in the level of proteolytic activity in prostatic cytosol fractions derived from mature animals, a change in the affinity of the receptor for oestradiol or an increase in oestrogen receptor levels in salt-extracted nuclear fractions. Administration of tamoxifen (1 mg/day) to intact guinea-pigs throughout the transpubertal growth phase did not influence the age-related decrease in cytosolic and nuclear oestrogen receptor levels. In contrast, the decrease in oestrogen receptor levels was prevented by castration. Administration of dihydrotestosterone (DHT; 1 mg/day) to intact prepubertal animals for 4 days before study resulted in diminished cytosolic oestrogen receptor levels; this effect of DHT was blocked by the non-steroidal antiandrogen flutamide (2 mg/day). Furthermore, elimination of testicular hormones by castration during the late-pubertal growth phase resulted in a greater than twofold increase in prostatic oestrogen receptor levels. Collectively, these observations suggest an age-related decrease in oestrogen receptor levels in the guinea-pig prostate which, in part, may be due to increased testicular function at puberty.

Effects of oestradiol-17 beta and 5 alpha-dihydrotestosterone on guinea-pig prostate smooth muscle cell proliferation and steroid receptor expression in vitro.

Smooth muscle cells (SMCs) are the major cellular component of the prostatic stroma. The aim of this study was to examine the effects of oestradiol-17 beta (OE2) and 5 alpha-dihydrotestosterone (DHT) on the proliferation of guinea-pig prostate SMCs in vitro. OE2 stimulated SMC DNA synthesis at all concentrations examined. At a plating density of 3.0 x 10(4) cells/cm2, maximal incorporation of [3H]thymidine (136% of control) was observed after 36 h of treatment with 1 nmol OE2/l. At the same plating density, DHT had an inhibitory effect on SMC DNA synthesis, with maximal effects (73% of control) being observed 24 h after treatment with 1 nmol DHT/l. These effects of OE2 and DHT were prevented by co-incubation with specific steroid receptor antagonists. At a threefold lower plating density (1.0 x 10(4) cells/cm2), the maximal stimulatory and inhibitory effects of OE2 and DHT were delayed by approximately 24 and 12 h respectively. At the lower plating density, a biphasic effect of DHT was observed on DNA synthesis; DHT was both inhibitory and stimulatory. Maximal inhibition (71% of control) and maximal stimulation (168% of control) were observed after 36 and 134 h treatment with DHT respectively. At the lower plating density, longer term treatment of SMC cultures with OE2 and DHT also resulted in an increase in cell number. After 7 days of treatment with OE2 and DHT, cell number increased by 13% and 12% respectively. When OE2 and DHT were added in combination, the short-term inhibitory effect of DHT on SMC DNA synthesis was dominant over the stimulatory effect of OE2. Treatment with DHT for 24 h significantly inhibited OE2-induced stimulation of [3H]thymidine incorporation, irrespective of the prior duration of OE2 treatment. At the lower plating density, OE2 also decreased oestrogen receptor (ER) mRNA levels to 38% of control levels after 24 h of treatment. ER mRNA levels remained repressed until 72 h after treatment with OE2, and returned to control values following 96 h of treatment. Both the androgen-induced inhibition and stimulation of DNA synthesis observed following treatment of SMCs with 1 nmol DHT/l were associated with a reduction in androgen receptor (AR) mRNA levels. At an intermediate time (i.e. 48 h after commencement of treatment with DHT) AR mRNA levels were increased more than twofold over control levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgen receptor agonist activity of the synthetic progestin, medroxyprogesterone acetate, in human breast cancer cells.

Medroxyprogesterone acetate (MPA), which is frequently used as second line hormonal therapy for the treatment of metastatic breast cancer, binds with high affinity to the progesterone receptor (PR). However, the androgenic side-effects of MPA suggest that it may also activate androgen receptor (AR) regulated pathways. Treatment of the human breast cancer cell lines MDA-MB-453, ZR-75-1 and T47-D with high dose (100 nM) MPA resulted in 26-30% inhibition of cell growth, which was partially reversed by co-treatment with a 10-fold excess of the synthetic antiandrogen, anandron. Scatchard analysis demonstrated specific, high affinity (non-PR) binding of [3H]MPA to cytosols prepared from the PR-/AR+ MDA-MB-453 and PR+/AR+ ZR-75-1, but not the PR-/AR- BT-20 breast cancer cell lines. Competition of [3H]MPA binding to MDA-MB-453 cytosols by equimolar concentrations of androgens (5alpha-dihydrotestosterone (DHT), R1881) and the antiandrogen, anandron was consistent with binding of MPA to the AR. In ZR-75-1 cell cytosol fractions, DHT, R1881 and anandron only partially competed out [3H]MPA binding, suggesting that androgens displace [3H]MPA binding to AR but not to PR. Induction by MPA of AR transactivation was demonstrated in MDA-MB-453 and ZR-75-1 cells, and in the CV-1 cell line transfected with a full-length AR. In these cell lines the increased activity of the androgen responsive reporter gene (MMTV-CAT) by 1 nM MPA was fully (MDA-MB-453, CV-1) or partially (ZR-75-1) inhibited by co-culture with 1 microM anandron. These findings indicate that MPA is an AR agonist and suggest that the in vivo effects of MPA in breast cancer patients may in part be mediated by the AR.

Androgens induce divergent proliferative responses in human breast cancer cell lines.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

Evidence for a novel mechanism of androgen resistance in the human prostate cancer cell line, PC-3.

Progression to hormone-refractory disease is a common outcome of human prostate cancer. In this study, we have investigated the basis of androgen insensitivity in the human prostate cancer cell line, PC-3, which was derived from a bone metastasis of a hormone-refractory prostate cancer. PC-3 cells with virtually undetectable (PC-3AR-) or low (PC-3AR+) levels of androgen receptor (AR) RNA expression were examined. RNase protection assays demonstrated that the level of AR RNA in PC-3AR+ cells was similar to that in a normal androgen-responsive genital skin fibroblast cell strain. Quantitative immunocytochemical staining of AR in PC-3AR+ cells using antibodies directed against the amino and carboxyl termini of the receptor revealed staining in 30% of cells with either antibody. Furthermore, the level of AR staining in PC-3AR+ cells was higher than in the androgen-responsive breast cancer cell lines ZR-75-1, T47-D, and MCF-7. Despite the expression of AR RNA and protein, PC-3AR+ cell proliferation was unaffected by the presence of 0.1-10 nM mibolerone. Scatchard analysis demonstrated a complete absence of specific [3H]dihydrotestosterone ([3H]DHT) binding to PC-3AR+ cytosolic extracts, which could not be explained by structural alterations in the AR gene. The sizes of individual AR exons amplified from genomic DNA derived from PC-3AR+ cells were identical to those amplified from normal human cells. Furthermore, sequence analysis did not reveal a mutation in the DNA- or hormone-binding domains of the AR gene in PC-3AR+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor microvascularity has no independent prognostic significance for breast cancer.

There is a continuing controversy regarding the value of estimating degree of intra-tumor vascularity to predict prognosis in breast cancer. In order to resolve this controversy, primary tumors from a cohort of 519 women with breast cancer were analysed to determine whether association exists between degree of vascularity and prognosis. Tumor vascularity was estimated by immunohistochemistry using a monoclonal antibody to the antigen CD31. The tumor area showing the highest degree of vascularity was chosen to score the number of microvessels per unit area. Issues such as the reproducibility of the microvascularity score and its association with tumor parameters including size, histological grade and hormone receptor levels were investigated. Although previously agreed criteria were used, consensus between two pathologists' estimations of the degree of vascularity was only moderate. There was no statistically significant association between tumor vascularity score and other currently established parameters of prognosis. After a median follow up of 71 months for axillary node negative patients, there was no association between tumor vascularity score and increased risk of relapse or death from breast cancer. In axillary node positive patients, tumor vascularity score was associated with increased risk of relapse and death from breast cancer. This association was not however independent of other established parameters of prognosis.

A comparison of methods for measuring intestinal antibodies against Vibrio cholerae in mice.

A re-evaluation was made of the efficiency with which some of the commonly used assays would detect intestinal antibodies. The data indicate that the most sensitive assays for the detection of intestinal antibodies are the baby mouse protection test and the radioimmunoassay. The reasons for the lack of sensitivity with other assay methods are discussed.

Intestinal antibody to Vibrio cholerae in immunised mice.

The immune response of the mouse to priming and booster doses of V. cholerae was studied to establish whether serum antibody could be used as a correlate of local immunity. Serum antibody titres following oral boosting of orally-primed animals were shown to reflect the state of local intestinal immunity. This was not the case when the same oral booster dose was given to parenterally-primed animals. These results were discussed in relation to the human endemic situation. The highest titres of intestinal protective antibodies were found following combination of the oral and parenteral routes of immunisation. Various killed or extracted preparations of V. cholerae were used as oral vaccines to test their ability to induce protective antibodies in the gut. Only Boivin antigen was capable of inducing as good an intestinal antibody response as would the living organism.

Modifications of the local immune response to Vibrio cholerate attributed to the intestinal microbial flora of the mouse.

Oral immunisation studies in germfree, specific pathogen-free (SPF) and conventionalised mice illustrated that the autochthonous gut flora can have a suppressive effect on the induction of a local intestinal immune response to Vibrio cholerae. Temporary colonisation of the small bowel by viable vibrios occurred only in the germfree animal. The lack of colonisation in SPF and conventionalised mice was presumably a cause of their lower coproantibody responses. Prevention of colonisation was probably due to bacterial antagonism rather than to cross-reaction antibodies. This conclusion was reinforced by studies involving oral immunisation of SPF mice maintained on streptomycin, and of conventionalised ex germfree mice. In addition to the increased protective coporantibody response of animals with reduced gut flora, there were increased levels of non-complement-fixing protective antibodies in their serum, which were probably derived from the guy lamina propria.

The significance of oncogene amplification in primary breast cancer.

Alterations in the gene copy numbers of the proto-oncogenes HER2/neu and c-myc in primary human breast cancer investigated in 73 patients. We detected amplification of HER2/neu in 17 patient samples and amplification of c-myc in 11, while amplification of both was seen in 6 samples. There was no correlation of age, hormone receptor positivity or tumour size with amplification of either proto-oncogene. Amplification of HER2/neu was significantly correlated with the stage of the disease. HER2/neu amplification was observed in 18.5% and 38% of node-negative and node-positive patients, respectively; the association between HER2/neu amplification and advanced stage of the disease was statistically significant (p = 0.05). Since this is a prospective study, the clinical significance of oncogene amplification is not known. The relatively high frequency of HER2/neu amplification points to a functional role in human breast cancer, particularly in the progression of the disease. The method used in our study allows oncogene amplification to be studied in conjunction with hormone receptor determination and thus may be of value in large clinical trials to determine the significance of oncogene abnormalities in breast cancer.