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We used a mixture of antisera to prostatic specific acid phosphatase, prostatic specific antigen, epithelial membrane antigen and cytokeratin to examine multiple marrow aspirates from patients with local (15) and metastatic prostatic carcinoma (15), and benign prostatic hypertrophy (10). We found moderate to large numbers of tumor cells in the bone marrow of 11 of 15 (73 per cent) patients with known metastatic disease and small numbers of abnormal cells in 2 of 15 (13 per cent) patients with apparently local disease. No tumor cells were found in patients with benign prostatic hypertrophy, and only 2 patients with metastatic disease had tumor cells in the bone marrow when conventional hematomorphological preparations were examined. These findings suggest that immunocytochemistry can increase the detection rate of metastatic prostatic carcinoma cells. Further followup of larger numbers of patients with local carcinoma will reveal whether the presence of micrometastases denotes a poor prognosis.

A variety of monoclonal antibody probes to the human breast have been prepared by various groups of workers. These probes are revealing hitherto unrecognized biological and biochemical differences in both normal and neoplastic breast cells. Further study is needed, however, in order to understand fully the significance of these differences. In the meantime, the use of immune probes can improve our ability to detect disease at an earlier stage in sites such as the bone marrow, and may be heralding a new era in the detection and treatment of disseminated breast cancer.

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.

By means of an immunocytochemical method the 1.25-dihydroxyvitamin D [1.25(OH)2D] receptor status of tumours from 136 patients with primary carcinoma of the breast was determined. Patients with receptor-positive tumours had significantly longer disease-free survival than those with receptor-negative tumours (Chi2 = 4.01, p less than 0.05). 1.25(OH)2D3 inhibits the proliferation of several established human breast cancer cell lines in vitro. Effects of 1.25(OH)2D3 on breast tumour growth in vitro were assessed by means of the nitrosomethylurea-induced rat mammary tumour model of hormone-responsive breast cancer. Treatment of tumour-bearing animals with 0.1 microgram of the synthetic analogue, 1 alpha-hydroxyvitamin D3, three times weekly produced significant inhibition of tumour progression. Taken together, these studies suggest that the levels of 1.25(OH)2D occurring in vivo may exert an inhibitory effect on receptor-positive tumours. Further studies are required to evaluate the role of vitamin D metabolites in the treatment of human malignant disease.

Factors influencing the extremes of survival in disseminated breast cancer were analysed. From a pool of 1066 patients with distant metastatic breast cancer, the 41 patients who had survived 5 yr or more from first distant recurrence were matched with the 41 patients with the shortest survival times. Both groups were compared with the pool and, where relevant, with each other. There were no differences with respect to presentation clinical stage, histological subtype, adjuvant therapy or the likelihood of menopause during the disease-free interval. There were significant differences in age, menopausal status, primary treatment, grade, pathological node involvement, time to recurrence, situation and number of sites of metastatic involvement, and response to treatment. The interpretation of these relationships as causal or casual is uncertain; however, given the magnitude of the survival differences, we expected the associated differences to be much greater than we found.

Frozen sections of breast cancer specimens from 114 patients were assayed for the presence of estrogen receptor (ER) with an immunocytochemical method (ERICA). Significant positive correlations were found between receptor status and various pathologic features, including better histologic degrees of differentiation (P less than 0.001), smaller tumor cell size (P = 0.001), and lower levels of either tumor necrosis (P less than 0.001) or lymphocytic infiltration (P less than 0.001). There was a significant relationship between tubular formation and ER positivity (P = 0.003). This study confirms the results from some previous studies in which ER, assessed by biochemical means, has shown a correlation with good-prognosis pathologic features.

Three hundred and twenty-two postmenopausal patients with primary breast cancer and ipsilateral axillary node involvement were randomized to receive aminoglutethimide and hydrocortisone or placebo for 2 years in a double blind randomized trial between April 1980 and March 1985. Two hundred and eighty-six patients were eligible for the study of whom 145 received active drug and 141 received placebo. At the present time significantly fewer patients have relapsed or died without previous relapse in the treatment arm (P = 0.002); 43 of 145 (30%) patients receiving aminoglutethimide have relapsed or died compared with 63 of 141 (40%) of those receiving placebo. Local recurrence is also significantly reduced (P = 0.002) since only 6 patients receiving active treatment developed local recurrence compared to 21 receiving placebo. Side effects were severe enough to necessitate complete withdrawal or reduction of therapy in 27 of 145 (19%) in the treatment arm of the study compared with 21 of 141 (15%) in the placebo arm. A single treatment-related death occurred, due to agranulocytosis. Aminoglutethimide and hydrocortisone therefore delay relapse after surgery for primary breast cancer in postmenopausal women. It is too early to assess any effect on overall survival.

Fifty heavily pretreated postmenopausal patients with advanced breast cancer were treated with oral medroxyprogesterone acetate (1 g) daily for 1-18 months. Eight patients (16%) responded to treatment for periods up to 18 months and in an additional eight patients (16%) the disease stabilized for up to 10 months. Thirty-two patients (64%) developed adverse effects and eight (16%), all of whom had extensive pulmonary metastases, died a respiratory-related death thought possibly to have been brought on or accelerated by treatment. Only eight patients (16%) experienced increased appetite, weight gain, feeling of wellbeing, or improved performance status. We conclude that medroxyprogesterone acetate is useful in patients with advanced metastatic disease but should be restricted to carefully selected patients and used only with extreme caution in patients with underlying cardiovascular or respiratory disease. Its major application probably lies in the benefit it confers earlier in the disease.

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.

Thirty-one post-menopausal female patients, with locally advanced or disseminated breast cancer were treated with the aromatase inhibitor 4-hydroxyandrostenedione given orally at a dose of 500 mg daily. Twenty-nine patients had assessable disease. Eight patients (28%) had objective evidence of partial response and six remain in remission 7-10 months later. A further four patients (14%) had stabilisation of disease and 11 patients (37%) had progressive disease in spite of treatment. Plasma oestradiol levels were measured throughout therapy in 16 patients and were lowered to 53% +/- 8% of baseline levels within 7 days of commencing 4-hydroxyandrostenedione. With regard to toxicity, one patient developed a transient skin rash and another patient some facial swelling. A further patient developed a transient leucopaenia and treatment was therefore discontinued. Twenty-seven of the 30 evaluable patients (90%) experienced no side effects. These results indicate that oral administration of 4-hydroxyandrostenedione is an acceptable new treatment for post-menopausal women with disseminated breast cancer.

4-hydroxyandrostenedione, a potent inhibitor of the aromatase (oestrogen synthetase) system, was given to 11 patients with metastatic breast cancer. After a single 500 mg intramuscular injection a sustained reduction of serum oestradiol was observed for at least 1 week in all patients in whom the steroid was measured. 4 patients responded to treatment for periods of up to 4 months, and healing of bone metastases and reduction in size of soft-tissue metastases was evident. The only side-effects were pain at the injection site and hot flushes. 4-hydroxyandrostenedione is a new and specific aromatase inhibitor which shows promise in the treatment of patients with metastatic breast cancer.

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.

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.

The immunohistochemical analysis of human beta- and kappa-caseins in the human breast, benign breast disease, and breast carcinomas is reported. The monoclonal antibodies LICR-LON-32.2 and LICR-LON-14.1 which react with human beta- and kappa-casein, respectively (Earl, H. M., and McIlhinney, R. A. J. Mol. Immunol., 22: 981-991, 1985) were used for these studies. Human beta- and kappa-caseins were detected in lactating human breast tissue, lactational foci in the resting breast, and heterogeneously in the 16th-week pregnant breast, but not in normal breast tissue. In benign breast disease occasional epithelial cells were demonstrated to synthesize beta- and kappa-caseins, but this finding did not appear to correlate with the hormonal status or previous obstetric histories of the patients. However, similar studies in 45 breast carcinomas with a wide range of estrogen receptor content, demonstrated no detectable beta- or kappa-casein. These results demonstrate that the caseins, which are biochemical markers of mammary gland differentiated function, and have been previously put forward as markers of breast cancer, were not synthesized in the 45 human breast carcinomas studied here, even in tumors with high levels of estrogen receptor protein.

We have treated 128 postmenopausal women with advanced breast cancer with 4-hydroxyandrostenedione. Of these, 118 were assessable for toxicity and 100 for response to treatment. Response to therapy was seen in 34% of patients and stabilization of disease in 12 patients. Three dose regimens were used (500 mg intramuscularly weekly; 250 mg intramuscularly every 2 weeks; and 500 mg orally daily). There was no difference in response in these three groups. Side effects were minimal and local reaction to injected drug was seen in 13% of patients. The sole severe side effect observed was neutropenia which was transient and reversible on discontinuing therapy. 4-Hydroxyandrostenedione is an effective nontoxic agent in the treatment of breast cancer.

Single cells from mammary carcinoma infiltrating bone marrow can be detected in marrow aspirates using immunocytochemical stains for epithelial membrane antigen (EMA). This technique has been used to examine marrow aspirates taken from multiple sites from 24 patients at surgery for breast cancer. Ten of these patients had EMA-positive cells in their marrow, while 32 marrow samples from patients who did not have carcinoma were negative. These results have been combined with those obtained by taking aspirates from single sites from 47 breast patients without known skeletal deposits. Follow up showed that the patients with EMA-positive cells in their marrow developed bone metastases at a significantly faster rate.

The value of a monoclonal antibody, LICR-LON-Fib-75 (Fib-75), which mediates complement lysis and recognises an antigen present on all epithelial-tumour cells so far studied but not on lymphoid cells or bone-marrow stem cells, in clearing infiltrated bone marrow was assessed. Chromium-51-release and trypan-blue-exclusion assays showed that no tumour cells were viable after exposure to Fib-75 and complement except when large clumps (greater than 500 cells) were present, whereas Fib-75 had no significant effect on bone-marrow cells, as judged by colony-forming-unit and pluripotential-stem-cell assays.

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.

Immunoreactive calcitonin was found in extracts of seven out of eight consecutive breast carcinomas and four selected lung carcinomas, but not in extracts of benign breast lesions or normal tissues. This suggests that the high plasma calcitonin levels observed in patients with a wide variety of cancers reflect ectopic production of calcitonin by cancer tissue, and that the radioimmunoassay for calcitonin could prove to be of value in the detection and management of malignant disease, particularly breast cancer.

N-Methyl-N-nitrosourea (MNU) given iv to rats 50-55 days old induced mammary tumors in 70% of F344/N and 91% W/ICRF inbred females with mean latency periods of 149 and 93 days, respectively. Reduction of the MNU dose did not affect tumor incidence in W/ICRF rats. Of the mammary tumors, 98% were classified histologically as adenocarcinomas, which grew progressively. Primary tumors of nonmammary origin were detected at low incidence. Upon histologic examination, no evidence was found for metastases of either the mammary or other primary tumors. No evidence for tumor-induced hypercalcemia was found. Oophorectomy at the time of MNU administration prevented tumor development; oophorectomy when at least 1 tumor/animal was palpable caused growth delay or regression. All MNU-induced and 7,12-dimethylbenz[a]anthracene-induced mammary tumors tested contained cytoplasmic estrogen receptor (ER) at similar concentrations and were indistinguishable histologically. MNU-induced tumors in F344 rats were transplantable and retained ER through three transplantations.

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 micrograms 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.

Monolayer tissue culture has been used as a system in which to study aspects of ectopic hormone secretion. Of a series of twenty-four human bronchial carcinomas, nineteen were successfully established in culture and the supernatant medium from each tested for peptide hormones by radioimmunoassay. Six tumours were found to produce adrenocorticotrophin (ACTH), four to release calcitonin (CT) and one to release both of these hormones. No growth hormone or insulin was detected throughout the series. Net in vitro synthesis of both ACTH and CT was demonstrated by recovery of more hormone during culture than was originally contained in the explanted tumour tissue. The production of hormone by four out of six proliferative cultures established, and its persistence through many subculture passages, confirms ectopic hormone production as a stable heritable characteristic of some lung tumours. The ability of hormone-producing bronchial tumour cells to respond to factors known to influence hormone output from normal endocrine cells was tested. ACTH release was stimulated in one tumour by Pitressin and CT in another by gastrin. In addition, the release of CT from the same tumour cell line was shown to be inhibited by the accumulation of high external concentrations of CT as has been reported for normal C-cells.

The importance of estrogen receptor (ER) determination in breast cancer is well established. Approximately 70% of ER-positive tumors are hormone responsive compared to 5-10% of ER-negative tumors. However, one-third of ER-positive tumors fail to respond, and the reasons for this are unclear. To further investigate these relationships we have determined levels of ER protein and mRNA in a number of human breast cancer biopsies. ER protein was estimated by the dextran-coated charcoal steroid binding method and by an ER immunocytochemical assay using a specific monoclonal antibody. A complimentary DNA clone (lambda OR3) encoding part of the human ER was used to determine mRNA levels. Dot blot analysis of twenty-seven tumors revealed a close agreement between ER mRNA and the dextran-coated charcoal assay (rs = 0.9; P less than 0.001). ER immunocytochemical assay staining also correlated with ER mRNA in twenty-five cases (rs = 0.75; P less than 0.001). Tumors from postmenopausal patients contained much higher levels of ER mRNA and ER protein than their premenopausal counterparts. ER-negative tumors produced no measurable ER mRNA. Northern blot analysis revealed a 6.4- and 3.7-kilobase species in ER-positive tumors and also in the human breast cancer cell line MCF-7. No differences in transcript sizes were found in tumors from hormone-responsive patients compared to nonresponding patients. We have also demonstrated, in tissue sections of normal and malignant breast, localization of ER mRNA by in situ hybridization to the same population of cells which exhibit immunoreactive ER.

Using a rat monoclonal antibody raised against human progesterone receptor (PR) we have developed an immunocytochemical technique to detect PR in human normal and malignant breast tissue and have compared the distribution of this with that obtained by the conventional dextran-coated charcoal steroid-binding assay. Immunoreactive PR was detected exclusively in the nuclei of epithelial cells in 29/51 (56.9%) of breast cancers studied. There was an excellent correlation between the immunocytochemical and dextran-coated charcoal techniques, with concordance in 43/51 (84.3%) cases [regression coefficient (Spearman) = 0.78; P less than 0.001]. The relationship between PR and estrogen receptor (ER) was also examined immunocytochemically using a monoclonal antiserum to ER. Twenty-eight out of 51 (54.9%) tumors were positive for both receptors and 13/51 (25.5%) negative for both. ER-positive, PR-negative tumors were found in 9/51 (17.6%) cases whereas only one case (2%) was PR-positive, ER-negative.

We have developed an immunocytochemical staining procedure (ERICA) using a monoclonal antibody to the estrogen receptor (ER) to determine ER status from samples obtained by fine needle aspiration of primary and recurrent breast cancer tissue (cyto-ERICA). ER status was assessable on 214 of 246 smeared aspirates from breast cancer patients. In 143 (66.8%) assessable smears positive nuclear staining was observed but was completely absent in 71 (33.2%) cases. In 107 cases we were able to compare results with those obtained with the quantifiable dextrancoated charcoal (DCC) radioligand binding technique using surgically excised material. We observed qualitative agreement in 53 of 62 (85.5%) of primary specimens and 16 of 16 (100%) recurrent samples compared to the subsequent DCC result on the same sample. Aspirates obtained from new secondary deposits were also assessed and in 16 of 19 (84.2%) cases results agreed with that established previously by DCC on the primary breast tumor. In a further 6 of 10 (60%) cases the cyto-ERICA result obtained from recurrent samples qualitatively agreed with that determined by DCC on a previous recurrent lesion. A comparison of staining of aspirates was also made against frozen tissue sections stained with the monoclonal antibody (tissue-ERICA). Where comparison was made of primary tumor specimens agreement was observed in 40 of 45 (88.9%) of cases while specimens from secondary lesions agreed qualitatively in 14 of 17 (82.3%) of cases. In a small number of samples where tissue-ERICA was performed on an earlier lesion to that aspirated for cyto-ERICA an agreement of 4 of 5 (80%) was observed. This technique shows good sensitivity in demonstrating ER in aspirate specimens, should therefore permit us to determine ER status before surgery for primary breast cancer, and may also mean that surgery for recurrent disease to determine receptor status is no longer necessary.

Of ten tumour markers measured every 3 months after mastectomy in patients with apparently localised primary breast cancer, plasma levels of alkaline phosphatase, carcinoembryonic antigen, and gamma-glutamyl transpeptidase were the most useful in detecting metastatic disease. With these three tests a "lead interval" of 3 months or more was obtained in about half the 23 patients who developed overt metases. Clinical examination, chest X-ray, and these three markers proved the most useful combination of tests in screening for metastases, since at least one test was abnormal in 46 of 47 patients at the time of the development of metastases as judged by more detailed physical tests.

Derivatives of tamoxifen (1) and 4-hydroxy-2-methyltamoxifen (2) in which the basic side chain has been modified by N-oxidation or by quaternization have been investigated with respect to the effects on affinity for the oestrogen receptor and on cytostatic activity towards the MCF-7 cell line in vitro. In addition to the conventional cytosol assay for receptor binding affinity (RBA) a recently developed whole-cell assay was employed. N-oxidation (e.g. 2----3) produced no significant alteration in RBA value either in cytosol or in whole cells, nor in activity towards the MCF-7 line. Quaternization with methyl iodide (1----4, 2----6) or ethyl bromide (1----5, 2----7b: the cis isomer 7a also formed) did not alter receptor binding in the cytosol assay but almost abolished binding in the whole cell and cytostatic activity. The whole-cell RBA values for 2 (0.45) and 3 (0.5) were lower than those for 4-hydroxytamoxifen (2.9), suggested to be due to the lower oestrogenicity of the 2-methyl derivatives since activity against MCF-7 cells was unimpaired. The even lower values of whole-cell RBA (0.01-0.02) for the quaternary ethyl bromide derivatives 7a and 7b were ascribed to poor penetration into the cell since these compounds had minimal cytostatic activity.

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.

4-Hydroxyandrost-4-ene-3,17-dione (HAD) is a potent and selective inhibitor of the enzyme complex aromatase, both in vitro and in vivo. The glucuronide is a major metabolite in the urine of patients and in the bile of rats given HAD and it was identified by chemical ionization-MS of the permethylated derivative. HAD glucuronide was quantified by first converting it enzymically into HAD, then determining HAD by capillary column GC-MS of the perfluorotolyl derivative using 4-hydroxyandrost-2,4-diene-3,17-dione as internal standard.

Of 1116 patients receiving primary treatment for breast carcinoma at the Royal Marsden Hospital since 1976, 651 had an abnormal bone scintigram either at primary diagnosis (378) or on subsequent follow-up (273) and 167 developed radiographically detectable bone metastases (21 at the time of primary diagnosis). Comparison of bone scintigrams and X-rays showed that scintigraphy was an inaccurate localiser of existing radiographic detectable metastases. If X-rays alone are used to detect bone metastases a limited examination with five plates will detect metastases with 92% accuracy. After primary surgery, routine X-ray screening for bone metastases is not necessary since it is possible to identify patients at risk on the basis of clinical examination, chest X-ray, and serum alkaline phosphatase and gamma-glutamyl transpeptidase levels.

Patients with breast cancer and bone destruction were found to have a pattern of calcium metabolism which was broadly similar to that found in other malignancies, but different from that in primary hyperparathyroidism. Thus, they tended to have reduced absorption of calcium from the intestine, elevated endogenous faecal calcium and normal or reduced urinary cyclic AMP excretion. Since prostaglandin synthetase inhibitors have been shown to inhibit breast cancer-induced osteolysis in vitro we have attempted to reduce bone destruction and serum calcium in patients with hypercalcaemia complicating breast cancer using these agents. High doses failed to reduce the serum calcium or the urinary hydroxyproline: creatinine ratio in ten patients with skeletal metastases, four of whom had hypercalcaemia.

A young man presented with rapidly developing Cushing's syndrome which was due to the ectopic secretion of ACTH and beta-MSH-like material from hepatic tumour deposits, possibly originating from biliary radicals. This association of the ectopic ACTH syndrome has not previously been described. During the 22 month course of the illness the plasma immunoreactive ACTH and 'beta-MSH' concentrations rose by logarithmic progression. The plasma calcitonin concentration was also raised but did not change during the last 12 months. At any stage of the illness the plasma concentration of the ecotopically produced hormones was stable except that after hydrocortisone there were inconstant variations. During the course of the illness the ectopic ACTH became biologically less potent. This ineffectural ACTH was present in the plasma, in the tumour, and in the medium in which the tumour was cultured, in a large molecular weight form. This 'big ACTH' differed from the normal ACTH found in the patient's pituitary and from authentic ACTH in its immunochemical character: the C-terminal antigenic determinant (33-39 region of ACTH) was masked in the large molecular weight form but was uncovered after extraction in neutral buffer and this 'big ACTH' was more readily extracted from the tumour at pH 7.0. The tumour tissue also contained immunoreactive beta-MSH-like material and immunoreactive calcitonin which resembled calcitonin M chromatographically.

15 patients receiving cytotoxic drugs (other than cisplatin) that had previously produced nausea and vomiting refractory to first-line antiemetics were given a selective 5-HT3 receptor antagonist (GR38032F), 4 mg intravenously and 4 mg orally, immediately before chemotherapy, the oral dose being repeated 5 and 10 h later. Nausea, vomiting, and side-effects were recorded for the ensuing 24 h. The 15 patients received a total of thirty-one courses of chemotherapy. Only 1 patient vomited. The only adverse events were dryness of the mouth in 1 patient, mild sedation in 1, and diarrhoea in 1, and these were not clearly drug related.

Using an immunocytochemical technique, micrometastases have been found in the bone marrow of approximately 26% of patients with primary breast cancer at the time of initial surgery. To determine the fate of these cells, both in patients receiving and not receiving adjuvant therapy, multiple bone marrow aspirates were repeated in 82 patients at a median time of 18 months after surgery but prior to overt relapse. In both treated and untreated patients micrometastases were only found in one of 45 (2%) and one of 37 (3%) patients, respectively. However, when multiple marrow aspirates were taken from patients with local recurrence the incidence of micrometastases was 19% (three of 16), and this increased to 30% (three of ten) in patients with disease at distant sites other than bone, and 100% (ten of ten) in patients with radiologically proven bony disease. Three of 11 (27%) patients in whom the primary tumor remained in situ while receiving adjuvant therapy before definitive surgery had micrometastases at the time of diagnosis and at follow-up 3 months later. These results suggest that many of the micrometastases from breast cancer patients are the result of "shedding" of cells from the primary carcinoma and that a proportion are not viable. The technique is currently insufficiently sensitive to accurately monitor adjuvant therapy in breast cancer patients.

A simple, reproducible extraction method has been developed, which is capable of measuring calcitonin in normal individuals. Normal calcitonin levels show a circadian variation, with a peak around midday and respond to known stimuli for calcitonin release.

A postal survey of current management of breast cancer among surgeons in Great Britain was carried out. The results showed that there is no consensus among surgeons about the treatment of primary breast cancer. Many different types of primary surgery are practised, and patterns of referral for postoperative radiotherapy vary widely. Adjuvant systemic treatment is given often by some surgeons and not at all by others. Few patients are entered into controlled randomised trials.

Inhibition of aromatase to reduce estrogen production by peripheral and ovarian tissue could be a useful approach to treating hormone-dependent breast cancer. Several C19, 17 keto steroids have been identified as aromatase inhibitors. The most potent of these cause rapid competitive inhibition followed by enzyme inactivation. Injections of the compounds caused inhibition of peripheral aromatization in monkeys. In rats, these treatments result in inhibition of ovarian aromatase and estrogen secretion, accompanied by marked regression of carcinogen(DMBA or NMU)-induced mammary tumors. To date, 60 postmenopausal patients with advanced metastatic breast cancer and unselected for the presence of estrogen receptors have been treated with once weekly injections of 4-OHA. The mean estradiol level measured in 14 patients was significantly reduced to 36% of pretreatment values after 1 month and remained at this level for up to 4 months. There was no effect of treatment on gonadotropin levels. Although all patients had relapsed from previous therapy, complete or partial tumor regression occurred in 30% of patients while 15% had static disease. The results indicate that in these patients the responses are due to inhibition of peripheral aromatization and that 4-OHA may be of value in treating postmenopausal breast cancer.

Various methods have been evaluated for their ability to detect bone metastases in patients with breast cancer. Bone scanning and hydroxyproline measurements are insensitive and showed metastases in few patients with primary breast cancer despite the fact that most will develop bone metastases. We have therefore investigated the value of examining the bone marrow with immunocytochemical staining for breast carcinoma cells. Initial results in 68 patients with no evidence of bone metastases by conventional means indicated (a) that some patients have breast cancer cells in bone marrow despite having no evidence of dissemination using other tests, and (b) that patients with micrometastases relapse sooner than those patients with normal bone marrows.