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Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

FOXM1 has been implicated in taxane resistance, but the molecular mechanism involved remains elusive. In here, we show that FOXM1 depletion can sensitize breast cancer cells and mouse embryonic fibroblasts into entering paclitaxel-induced senescence, with the loss of clonogenic ability, and the induction of senescence-associated β-galactosidase activity and flat cell morphology. We also demonstrate that FOXM1 regulates the expression of the microtubulin-associated kinesin KIF20A at the transcriptional level directly through a Forkhead response element (FHRE) in its promoter. Similar to FOXM1, KIF20A expression is downregulated by paclitaxel in the sensitive MCF-7 breast cancer cells and deregulated in the paclitaxel-resistant MCF-7TaxRcells. KIF20A depletion also renders MCF-7 and MCF-7TaxRcells more sensitive to paclitaxel-induced cellular senescence. Crucially, resembling paclitaxel treatment, silencing of FOXM1 and KIF20A similarly promotes abnormal mitotic spindle morphology and chromosome alignment, which have been shown to induce mitotic catastrophe-dependent senescence. The physiological relevance of the regulation of KIF20A by FOXM1 is further highlighted by the strong and significant correlations between FOXM1 and KIF20A expression in breast cancer patient samples. Statistical analysis reveals that both FOXM1 and KIF20A protein and mRNA expression significantly associates with poor survival, consistent with a role of FOXM1 and KIF20A in paclitaxel action and resistance. Collectively, our findings suggest that paclitaxel targets the FOXM1-KIF20A axis to drive abnormal mitotic spindle formation and mitotic catastrophe and that deregulated FOXM1 and KIF20A expression may confer paclitaxel resistance. These findings provide insights into the underlying mechanisms of paclitaxel resistance and have implications for the development of predictive biomarkers and novel chemotherapeutic strategies for paclitaxel resistance.

Oestrogen receptor-α (ERα) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERα activity. In vitrostudies have indicated that phosphorylation of ERα, in particular on serine 118 (S118), can result in activation in a ligand-independent manner, thereby potentially contributing to resistance to endocrine agents, such as tamoxifen and aromatase inhibitors. Here we report the immunohistochemistry (IHC) of S118 phosphorylation in 301 primary breast tumour biopsies. Surprisingly, this analysis shows that S118 phosphorylation is higher in more differentiated tumours, suggesting that phosphorylation at this site is associated with a good prognosis in patients not previously treated with endocrine agents. However, we also report that S118 phosphorylation was elevated in tumour biopsies taken from patients who had relapsed following tamoxifen treatment, when compared to pre-treatment biopsies. Taken together, these data are consistent with the view that S118 phosphorylation is a feature of normal ERα function and that increases in levels of phosphorylation at this site may play a key role in the emergence of endocrine resistance in breast cancer.

The histone deacetylase inhibitors are a new class of cytostatic agents that inhibit the proliferation of tumor cells in culture and in vivo by inducing cell cycle arrest, differentiation and / or apoptosis. Histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription. Histone deacetylase inhibition induces the accumulation of hyperacetylated nucleosome core histones in most regions of chromatin but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes. Non-histone proteins such as transcription factors are also targets for acetylation with varying functional effects. Acetylation enhances the activity of some transcription factors such as the tumor suppressor p53 and the erythroid differentiation factor GATA-1 but may repress transcriptional activity of others including T cell factor and the co-activator ACTR. Recent studies in our laboratory and others have shown that the estrogen receptor α (ERα) can be hyperacetylated in response to histone deacetylase inhibition, suppressing ligand sensitivity and regulating transcriptional activation by histone deacetylase inhibitors. Conservation of the acetylated ERα motif in other nuclear receptors suggests that acetylation may play an important regulatory role in diverse nuclear receptor signaling functions. A number of structurally diverse histone deacetylase inhibitors have shown potent antitumor efficacy with little toxicity in vivo in animal models. Several compounds are currently in early phase clinical development as potential treatments for solid and hematological cancers both as monotherapy and in combination with cytotoxics and differentiation agents. This report reviews the biology and clinical development of histone deacetylase inhibitors for cancer therapy.

Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.

Over the past decade several novel aromatase inhibitors have been introduced into clinical practice. The discovery of these drugs followed on from the observation that the main mechanism of action of aminogluthemide was via inhibition of the enzyme aromatase thereby reducing peripheral levels of oestradiol in postmenopausal patients. The second-generation drug, 4-hydroxyandrostenedione (formestane), was introduced in 1990 and although its use was limited by its need to be given parenterally it was found to be a well-tolerated form of endocrine therapy. Third-generation inhibitors include vorozole, letrozole, anastrozole and exemestane, the former three being non-steroidal inhibitors, the latter being a steroidal inhibitor. All are capable of inhibiting aromatase action by >95% compared with 80% in the case of 4-hydroxyandrostenedione. The sequential use of different generations of aromatase inhibitors in the same patients is discussed. Studies suggest that an optimal sequence of these compounds may well result in longer remission in patients with hormone receptor positive tumours.

We have investigated the role of biochemical markers in breast and lung cancer but have found that currently available tests have little role in management. In breast cancer, for example, we have found that the most sensitive method for routine screening for metastases is by using chest x-ray and clinical examination, liver function tests, and carcinoembryonic antigen measurements. We are now examining other methods for detecting metastases using immunocytochemistry in cytological preparations of bone marrow and attempting to raise monoclonal antibodies to breast cancer cells. In lung cancer, the major effort has been directed towards characterisation of large-molecular-weight ectopic hormones, particularly calcitonin.

Hydroxylaminoglutethimide (3-ethyl-3-(4-hydroxylaminophenyl)-2,6-piperidinedione) has been identified as a novel metabolite of aminoglutethimide (3-(4-aminophenyl)-3-ethyl-2,6-piperidinedione) in the urine of patients treated chronically with this drug. The metabolite was isolated by reverse-phase thin-layer chromatography, and characterized by comparison of its mass spectrum and chromatographic properties with those of the synthetic compound. Hydroxylaminoglutethimide is unstable; it is readily oxidized to nitroso-glutethimide and disproportionates in the mass spectrometer into this compound and aminoglutethimide. In none of four patients studied was the metabolite detected in the urine after the first dose of the drug. In one patient it appeared after the second dose and in two more within seven to eight days suggesting that its formation is drug-induced, and that it may be the metabolite responsible for the diminished half-life of aminoglutethimide during chronic therapy. The profile of metabolites from one patient, examined by high-performance liquid chromatography after the first dose and again after six weeks of therapy afforded evidence that the formation of hydroxylaminoglutethimide was at the expense of a major metabolite N-acetylamino-glutethimide. Hydroxylaminoglutethimide was not an induced metabolite in the rat.

The cloning of a novel estrogen receptor beta (denoted ERbeta) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930 and Mosselman, S., Polman, J. , and Dijkema, R. (1996) FEBS Lett. 392, 49-53). ERbeta is highly homologous to the "classical" estrogen receptor alpha (here referred to as ERalpha), has been shown to bind estrogens with an affinity similar to that of ERalpha, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERalpha and beta in gel shift assays. We show that DNA binding by ERalpha and beta are similarly affected by elevated temperature in the absence of ligand or in the presence of 17beta-estradiol and the partial estrogen agonist 4-hydroxy-tamoxifen. In the absence of ligand, DNA binding by ERalpha and beta is rapidly lost at 37 degrees C, while in the presence of 17beta-estradiol and 4-hydroxy-tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182, 780 does not "protect" human ERalpha (hERalpha) from loss of DNA binding at elevated temperature in vitro, it does appear to protect human ERbeta (hERbeta), suggestive of differences in the way ICI 182, 780 acts on hERalpha and beta. We further report that ERalpha and beta can dimerize with each other, the DNA binding domain of hERalpha being sufficient for dimerization with hERbeta. Cell and promoter-specific transcription activation by ERalpha has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERalpha and beta add greater levels of complexity to transcription activation in response to estrogens.

A single intravenous injection of recombinant human tumour necrosis factor (TNF) resulted in significant, but transient (24–48 hr) reductions in food intake and body weight, and increases in rectal temperature, resting oxygen consumption (VO2) and brown adipose tissue (BAT) thermogenic activity (mitochondrial GDP-binding). The increased VO2 was inhibited by β-adrenergic blockade (propranolol), and activation of BAT was prevented by denervation of the tissue. In adult (4-month old) animals, TNF induced greater reductions in food intake and body weight, caused general malaise and some fatalities, but did not significantly alter VO2 or BAT activity. However, the reduction in VO2 following β-adrenergic blockade was greater in TNF-treated rats and BAT activity was enhanced when compared to pair-fed controls. Injection of adult rats with gamma-interferon induced small changes in body weight and temperature which were slightly potentiated when injected with a low dose of TNF. The results indicate that TNF stimulates sympathetic outflow to BAT. This effect may be partly responsible for the increases in body temperature and metabolic rate associated with TNF treatment and with cancer cachexia.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are growth factors which may have a physiological role in the normal breast and in breast cancer. A study of the effects of aFGF and bFGF on a variety of breast cell lines and epithelial cells purified from normal breast organoids showed that whereas normal breast cells did not exhibit membrane ruffling in response to either of these growth factors, some breast cancer cell lines did. This difference was not due to lack of receptor since all the cell lines tested were mitogenically stimulated by bFGF. Dominant negative mutations of FGF receptor 3 (FGFR-3) and the small GTP-binding protein p21rac inhibited membrane ruffling, showing that receptor dimerization and phosphorylation and p21rac activation are prerequisites for membrane ruffling in response to aFGF and bFGF. Transient transfection of individual FGFRs into cos-7 cells showed that FGFR-1, FGFR-2 and FGFR-3 could not mediate a membrane ruffling response whereas FGFR-4 could. These studies elucidate one signalling mechanism of FGF and point to differences in the response of normal and cancer breast epithelial cells which may be important in cell motility.

The anti-tumour effect of EB 1089, a novel vitamin D analogue with reduced calcaemic activity, was examined in vivousing the N-methyl-nitrosourea-induced rat mammary tumour model. The vitamin D compound was given orally at a dose of 1 pg/kg body weight alone and in combination with tamoxifen (1 mg/kg). Effects were compared with oral tamoxifen treatment alone. EB 1089 significantly inhibited tumour progression compared with controls with a response rate of 58% and a regression rate of 92% As expected, tamoxifen at the dose given also caused significant inhibition of tumour progression with a response rate of 73%. Combination of these two compounds did not lead to a marked increase in their effectiveness. Histological examination of tumours from EB 1089-treated rats showed a marked reduction in cellularity and mitotic activity.At the dose given, EB 1089 produced a significant rise in serum calcium concentration and urinary calcium excretion. Tamoxifen treatment alone did not significantly alter serum calcium levels. However, combined treatment with tamoxifen and EB 1089 led to a significant reduction in hypercalcaemia compared with EB 1089 alone. It is suggested that vitamin D analogues with reduced calcaemic activity may provide a new therapeutic strategy for certain malignancies, either alone or in combination with established treatment regimens.Endocrine-Related Cancer(1996) 3327-335

Two assay procedures, an inhibition radioimmunoassay (Inhibition-RIA) and an immunoradiometric assay (IRMA), were established for the detection of circulating tumour-associated Ca antigen. There was a good correlation between results (r = 0.987) but the Inhibition-RIA was selected for extended tests on human sera from patients with breast disease because of its greater ease and economy in use. Circulating Ca antigen was not exclusive to malignancy and the level failed to discriminate between patients with primary carcinoma and those with benign disease. Ca antigen was present in sera of 100 healthy individuals (median 7.1 micrograms ml-1, range 1.8-24.4 micrograms ml)-1, 39 patients with benign disease (median 9.9 micrograms ml-1, range 2.5- greater than 100 micrograms ml-1) and in 67 patients with primary carcinoma (median 11.0 micrograms ml-1, range 3.8- greater than 100 micrograms ml-1). Elevated Ca antigen levels were found in 50% of patients with metastatic spread (median 30.7 micrograms ml-1, range 8.2- greater than 100 micrograms ml-1) and in some patients with primary disease but further studies are needed to determine the prognostic significance. Immunochemical studies confirmed that Ca antigen is a normal serum product but its function is unclear.

Serum prolactin concentrations were measured in 135 postmenopausal patients with advanced breast cancer prior to their treatment with one of 3 endocrine therapies: aminoglutethimide (AG), tamoxifen (T) + AG, or T + AG + danazol. The mean level of prolactin was higher, and there were more individuals with levels of prolactin greater than or equal to 500 mIUl-1, in the group of patients who did not respond to treatment. Of the patients whose disease progressed, those with prolactin levels greater than or equal to 500 mIUl-1 had a significantly shorter survival. It appears that high prolactin levels indicate a poor prognosis to endocrine therapy and the probability of a shorter than average survival time.

Gonadotrophins, oestradiol, androstenedione, testosterone and dehydroepiandrosterone sulphate (DHAS) were measured sequentially in 72 patients with advanced breast cancer receiving endocrine therapy of various types. Tamoxifen significantly reduced gonadotrophins but did not effect other hormones. Danazol also reduced gonadotrophins. Aminoglutethimide (AGT) reduced oestradiol and DHAS but had not effect on gonadotrophins. The effects of administering tamoxifen, AGT and danazol together (TAD) together were therefore examined. This combination reduced gonadotrophins, oestradiol and DHAS, but no further than tamoxifen and AGT alone. The degree and duration of hormone suppression were similar in both responders and non-responders to tamoxifen, AGT or TAD, though patients responding to AGT showed more complete suppression at the end of the course of treatment, perhaps because they were treated longer. On relapse, adequate gonadotrophin and steroid suppression was demonstrated in patients receiving tamoxifen and AGT respectively. We conclude that (a) response to endocrine therapy is unlikely to be related to the degree of endocrine suppression produced by the therapy, (b) combination endocrine therapy does not further reduce serum-hormone concentrations and (c) relapse is unlikely to be due to escape from the hormone-inhibitory effects of endocrine agents.

N-methyl-N-nitrosourea (MNU) given i.v. to female rats of inbred strains induces mammary adenocarcinomas which are hormone-sensitive but do not spontaneously metastasize (Williams et al., 1981). Tissue culture and selection techniques have been used to derive metastasizing tumours from a mammary tumour induced with MNU in F344/N rats. Histologically, primary tumours and metastases in the lung and lymph nodes were similar. These systems may constitute useful models for the study of breast-cancer metastasis.

We have studied separated human breast epithelial and myoepithelial cells for the presence of basic fibroblast growth factor (FGF2) and its receptors and for the effects of FGF2 on the proliferation of both cell types. We have also studied the role of activin and its receptor in controlling cell proliferation.Our results indicated that these cell types differ markedly in synthesis and response to FGF2 and activin and in their receptor content. FGF2 had no effect on the proliferation of myoepithelial cells but promoted the survival of the separated epithelial cells. Immunostainable FGF receptors 1 and 4 were present in epithelial cells and to a lesser extent in myoepithelial cells. These results indicated that myoepithelial-derived FGF2 may be important in controlling epithelial cell survival and that differential receptor expression could control FGF2 action in these different cell types.We found that activin β-a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we found only four (HBL-100, MCF-10A, PMC-42 and BT-20) to be positive for activin β-a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF-7 cells where it causes an arrest in G1. Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we have demonstrated that activin is an inhibitor of tubule formation by human mammary organoids in vitro.Endocrine-Related Cancer(1997) 435-43

The urinary excretion during 24 h of aminoglutethimide (AG) its major metabolite (N-acetylAG) and two minor metabolites (N-formylAG and nitroG) were measured in 10 volunteers given AG who had been typed for acetylator phenotype using sulphadimidine. The slow acetylators of sulphadimidine excreted more AG (mean 28% of the administered dose) than did the fast acetylators (12%), but the latter excreted more of the dose as N-acetylAG (8.8%) than did the former (3.9%). NitroG and N-formylAG were minor urinary metabolites of AG in humans. The former was more abundant in the urine of slow acetylators (0.10% of the dose) than in that of fast acetylators (0.047%), whereas the respective proportions of doses excreted as the N-formyl derivative (0.475 and 0.465%) were not significantly different for the two acetylator phenotypes. These results show that AG is among those drugs that are polymorphically acetylated in humans.

We have developed a technique for the immunocytochemical staining of marrow smears using antiserum to epithelial membrane antigen (EMA). This membrane component is confined to, but widely distributed in, epithelial tissues and tumours derived from them, and is strongly expressed by infiltrating breast carcinoma cells. Marrow aspirates from patients with both early and metastatic breast cancer have been examined, and the results of immunocytochemical staining compared with conventional cytology and histology. Staining with antiserum to EMA enabled us to detect small numbers of carcinoma cells, and increased the yield of positive samples. Furthermore, using this technique, we found malignant cells in the marrow of patients with primary breast cancer with no other evidence of metastatic disease. Thus immunocytochemical staining for EMA may be of value in the detection of micrometastases in patients with primary breast carcinoma.

We have investigated, by immunocytochemical means, the value of an antiserum raised to milk-fat-globule membranes in detecting metastatic deposits of breast carcinoma in conventional histological sections of liver, lymph nodes and marrow. The antiseum recognizes a membrane component, which we have called Epithelial Membrane Antigen, and which is confined to but widely distributed in epithelial tissues and tumours derived from them. In the sections examined, a greater extent of tumour infiltration was usually found, largely due to the identification of single malignant cells which normally go unrecognized with conventional stains. The number of positive samples was only increased, however, in sections of marrow aspirates, and the reasons for this are discussed. We suggest that further increases in detection rates could be attained by using the antiserum on cytological smears of marrow or even in cell suspensions.

Aspirin and indomethacin, flurbiprofen, synthetic human calcitonin, and mithramycin were assessed for their anti-osteolytic properties in patients with breast cancer and painful skeletal metastases.

A total of 23 patients with advanced malignancies received escalating doses of epirubicin (100–120 mg/m2) i.v. at 3-week intervals; 15 had received previous chemotherapy. In all, 46 courses of chemotherapy were given. Mucositis (grade II or III) occurred in 47% of courses at 120 mg/m2, but in only 15% of courses at 115 mg/m2. Myelotoxicity was manifest as leucopenia, with a median white blood count nadir of 1.9 (range, 0.8–7.0)×109/l. Nausea and vomiting were generally well controlled by prophylactic antiemetic therapy. Alopecia was WHO grade 0 in 2 patients, grade I in 1, grade II in 5 and grade III in 14. No renal or hepatic toxicity was noted, and there were no episodes of congestive cardiac failure. One fatal coronary thrombosis (proven at post-mortem examination) occurred 48 h after a dose of 115 mg/m2. Four patients developed thrombophlebitis at the injection site that was not dose-related; it occurred at doses between 100 and 120 mg/m2. Two patients who had been given chemotherapy in the past had complete responses (one penile carcinoma, one gastric carcinoma). Six patients had partial responses, including two with breast cancer, one with gastric cancer and three with sarcoma. Intermediate-dose epirubicin was well tolerated up to 120 mg/m2, when mucositis became a significant clinical problem. Preliminary data suggest promising activity in gastric cancer, breast cancer and a variety of sarcomas.

A single intravenous injection of recombinant human tumour necrosis factor (TNF) resulted in significant, but transient (24–48 hr) reductions in food intake and body weight, and increases in rectal temperature, resting oxygen consumption (VO2) and brown adipose tissue (BAT) thermogenic activity (mitochondrial GDP-binding). The increased VO2 was inhibited by ß-adrenergic blockade (propranolol), and activation of BAT was prevented by denervation of the tissue. In adult (4-month old) animals, TNF induced greater reductions in food intake and body weight, caused general malaise and some fatalities, but did not significantly alter VO2 or BAT activity. However, the reduction in VO2 following ß-adrenergic blockade was greater in TNF-treated rats and BAT activity was enhanced when compared to pair-fed controls. Injection of adult rats with gamma-interferon induced small changes in body weight and temperature which were slightly potentiated when injected with a low dose of TNF. The results indicate that TNF stimulates sympathetic outflow to BAT. This effect may be partly responsible for the increases in body temperature and metabolic rate associated with TNF treatment and with cancer cachexia.

The anti-tumour effect of EB 1089, a novel vitamin D analogue with reduced calcaemic activity, was examined in vivousing the N-methyl-nitrosourea-induced rat mammary tumour model. The vitamin D compound was given orally at a dose of 1 pg/kg body weight alone and in combination with tamoxifen (1 mg/kg). Effects were compared with oral tamoxifen treatment alone. EB 1089 significantly inhibited tumour progression compared with controls with a response rate of 58% and a regression rate of 92% As expected, tamoxifen at the dose given also caused significant inhibition of tumour progression with a response rate of 73%. Combination of these two compounds did not lead to a marked increase in their effectiveness. Histological examination of tumours from EB 1089-treated rats showed a marked reduction in cellularity and mitotic activity.At the dose given, EB 1089 produced a significant rise in serum calcium concentration and urinary calcium excretion. Tamoxifen treatment alone did not significantly alter serum calcium levels. However, combined treatment with tamoxifen and EB 1089 led to a significant reduction in hypercalcaemia compared with EB 1089 alone. It is suggested that vitamin D analogues with reduced calcaemic activity may provide a new therapeutic strategy for certain malignancies, either alone or in combination with established treatment regimens.Endocrine-Related Cancer(1996) 3327-335

We have studied separated human breast epithelial and myoepithelial cells for the presence of basic fibroblast growth factor (FGF2) and its receptors and for the effects of FGF2 on the proliferation of both cell types. We have also studied the role of activin and its receptor in controlling cell proliferation.Our results indicated that these cell types differ markedly in synthesis and response to FGF2 and activin and in their receptor content. FGF2 had no effect on the proliferation of myoepithelial cells but promoted the survival of the separated epithelial cells. Immunostainable FGF receptors 1 and 4 were present in epithelial cells and to a lesser extent in myoepithelial cells. These results indicated that myoepithelial-derived FGF2 may be important in controlling epithelial cell survival and that differential receptor expression could control FGF2 action in these different cell types.We found that activin β-a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we found only four (HBL-100, MCF-10A, PMC-42 and BT-20) to be positive for activin β-a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF-7 cells where it causes an arrest in G1. Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we have demonstrated that activin is an inhibitor of tubule formation by human mammary organoids in vitro.Endocrine-Related Cancer(1997) 435-43

Monolayer cultures have been established from a poorly differentiated carcinoma of the lung. Homogeneous cell growth and morphology have been maintained for over 18 months through more than 80 subculture passages, and the cells have been found to produce both immunoreactive calcitonin and an immunoreactive carcinoembryonic antigen-like material.

In order to determine the mechanisms of relapse following response to endocrine therapy, we have measured the oestrogen receptor (RE) content of biopsies of breast cancer in patients receiving various types of endocrine treatment. RE content fell in responding (means of 260.2 to 12 fmol/mg protein) and in nonresponding (means of 155.1 to 31.8 fmol/mg protein) patients who had measurable receptor at the start of treatment. Some of these patients, and a further group of responders to endocrine therapy, were monitored until relapse. Tumour biopsies at the time of relapse showed that 10/14 tumour samples contained significant RE (mean of 86.7 fmol/mg protein, range less than 10-271 fmol/mg protein) after successful endocrine therapy. No relationship could be found between RE content and plasma gonadotrophin or steroid-hormone concentration, but the fall in RE content correlated with reduced numbers of tumour cells in the biopsy. These results indicate that relapse following successful endocrine therapy in breast cancer does not appear to be due to the emergence of RE-negative tumour cells. The fall in RE content during response to endocrine therapy may be due to reduced tumour-cell content of the biopsy.

4-hydroxyandrostenedione is a steroidal, suicide substrate inhibitor of aromatase, which has been widely tested in postmenopausal breast cancer patients. It is highly specific with the only notable endocrine changes other than oestrogen suppression being a dose-related suppression of sex-hormone binding globulin when the drug is given orally (a reflection of the drug's minor androgenic activity). Intramuscular administration of 250 mg every second week is the schedule of choice. This achieves peripheral aromatase inhibition of about 85% and oestradiol suppression of about 65%. The drug is usually used second-line, after tamoxifen, with an overall response rate in unselected patients of 26%. Side-effects are minimal and consist almost entirely of local reactions at the site of injection. 4-hydroxyandrostenedione is therefore a useful new treatment option as the first selective aromatase inhibitor to have wide clinical availability.

In 141 postmenopausal node-positive patients with primary breast cancer, routine biochemical markers (alkaline phosphatase, gamma-glutamyl transpeptidase, carcinoembryonic antigen), and chest x-ray, in combination with history and clinical examination, have been performed at 3 monthly intervals for at least 2 years. Sixty one patients relapsed at a median time of 14 months. The recurrence was detected at routine follow-up in 40 (66%) patients. Of these 40 patients, 26 (65%) presented with symptoms, 11 (28%) were asymptomatic but were found to have relapsed on clinical examination, and only 3 (8%) had their relapse diagnosed on the basis of an abnormal chest x-ray. The remaining 21 patients presented early with symptoms. Therefore symptoms and clinical examination accounted for the detection of relapse in 58 of the 61 (95%) patients. Of the patients who had relapsed, 49% (30 of 61) had one or more abnormal markers/chest x-rays prior to relapse, rising to 79% (48 of 61) at the time of relapse. Of 80 patients with no evidence of recurrence, 36% (29) had no marker abnormality recorded, whereas in 64% (51) one or more abnormalities were found. These results suggest that history and examination are the important procedures in follow-up, and that abnormal markers are not always due to metastatic disease and may be misleading.

Computed tomography (CT) of bone was carried out in 20 patients with breast cancer, all of whom had abnormal radionuclide uptake on skeletal scintigrams but normal conventional radiographs. Twenty-eight sites were examined and 13 showed metastases in 11 patients. Five of these patients had no evidence of extra-skeletal recurrent disease. Follow-up at eight of these sites showed healing, sclerosis or progression, all of which correlated well with clinical findings.CT showed benign causes of radionuclide accumulation in three patients (7 sites) but no abnormality in six patients (8 sites). None of these patients has subsequently developed bone metastases.CT is superior to conventional radiographs in the diagnosis of skeletal metastases and should be carried out when skeletal scintigraphy is positive and conventional examinations are normal.

Repeated administration of long-acting analogues of gonadotrophin-releasing hormone down-regulates the pituitary gonadotrophins and gonadal hormones. The activity of these compounds in premenopausal women with breast cancer has been previously noted. In an attempt to cause a highly selective medical hypophysectomy 18 consecutive postmenopausal women with symptomatic advanced breast cancer were treated with intranasal buserelin in divided dosages of either 600 or 1000 µg daily. The pituitary gonadotrophins were suppressed in all patients, without objective evidence of response. This is in contrast with an earlier finding that the long-acting analogues of gonadotrophin-releasing hormone were effective in postmenopausal patients with breast cancer.

Fifty-seven patients with metastatic breast carcinoma have been treated with mitomycin C (10 mg/m2 IV 6-weekly), melphalan (6 mg/m2 POx3 days, 3-weekly), and methotrexate (35 mg/m2 IV 3-weekly) to assess the efficacy and toxicity of this regimen. Of 48 evaluable patients 19 (40%) responded for a median period of 5 months and 12 (25%) had stabilisation of disease. Of the 12 patients previously treated with adriamycin only one responded, whereas 18 of the 36 patients without previous chemotherapy responded. Although healing of bone metastases was infrequent control of hypercalcaemia was commonly seen. Generally the treatment was well tolerated and treatment was stopped in only five patients because of toxicity. Cummulative marrow toxicity was observed but was not a significant problem in the first 6 months of treatment. Mitomycin C, melphalan, and methotrexate (MMM) appears to provide an effective, well tolerated chemotherapy combination for metastatic breast carcinoma.

Pregnancy specific β1glycoprotein (SP1) was measured in plasma from patients with carcinoma of the breast (42 patients), lung (32 patients), and testis (9 patients). Elevated circulating levels of SP1were detected in one patient from each group.