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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.

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.

Numerous studies in man have shown that the assay of tumour markers in the blood and urine seldom yields sufficient forewarning of the development of metastatic disease. Accordingly, alternative approaches have been sought for their earlier detection. The deployment of immunological probes, both in vivo in the form of radioimmunolocalisation and in vitro using immunocytochemical methods at a histological level, is under active study. At present the latter method appears capable of detecting micrometastases in the bone marrow in subjects with breast cancer.

Many biochemical indices are purported to have clinical utility in the detection and management of neoplasia. Experience gained during the past decade tends to indicate their having a more important role in the detection and monitoring of metastases than of the primary lesion. From this present review of some of the commoner human tumours, it is concluded that such marker substances are important adjuncts in the management of germ cell and certain endocrine and endocrine-related tumours. The carcinoembryonic antigen (CEA) provides a marker for many gastrointestinal cancers, but there are no presently available substances with clinical usefulness for either breast or lung neoplasms. Alternative approaches to the detection of metastases are also presented. The particular use of antibody probes at an immunohistochemical level has been claimed to be able to detect micrometastastic disease in bone marrow or tumour-related monoclonal antibody probes may have application to other cancers in the future.

We have devised a method utilizing a monoclonal antibody-toxin conjugate (LICR-LON-Fib75/abrin A-chain) for ridding bone marrow of infiltrating breast cancer cells to rescue patients with autologous bone marrow following high dose therapy. Initially we examined the activity of this conjugate in vitro. Five of seven human breast cancer cell lines were killed following exposure at 10(-8) M for 2 h; this concentration only reduced bone marrow colony formation to 83% (range, 50-100%) of control bone marrow. We then examined the pattern of bone marrow recovery after high dose melphalan (200 mg/m2) in patients with advanced breast cancer who were in remission following combination chemotherapy. To do this we compared the time of recovery of the blood count in three patients who received treated marrow and seven who received untreated marrow. Mean time to recovery of the peripheral white count (greater than 1.5 X 10(9)/liter) was 16.7 days (treated) and 18.3 days (untreated), respectively. Mean time to recovery of peripheral platelet count (greater than 50 X 10(9)/liter) was 23.7 days (treated) and 18.9 days (untreated), respectively. Patients continued in remission for 1-greater than 14 mo after high dose melphalan, and remission duration was similar in patients who received treated (6.2 mo) and untreated (7.3 mo) bone marrow. These findings indicate that treatment of bone marrow with LICR-LON-Fib75/abrin A-chain conjugate does not significantly impair bone marrow recovery, and it is, therefore, possible to rescue breast cancer patients with bone marrow that has been cleansed of infiltrating cancer cells. This may have an application in patients with poor-risk primary breast cancer who have micrometastases and who may benefit from intensive therapy, but it has minimal application in patients with more advanced disease.

Many patients with advanced non-thyroid malignancies have elevated plasma immunoreactive calcitonin concentrations. Breast and bronchial carcinomas contain immunoreactive calcitonin and an epidermoid bronchial carcinoma has been shown to produce immunoreactive calcitonin in vitro. We have established monolayer cultures of breast carcinomas and eight out of fifteen consecutive carcinomas released immunoreactive calcitonin; some released HCG (human chorionic gonadotrophin) or CEA (carcinoembryonic antigen). In addition, a primary human breast carcinoma has been shown to release and contain calcitonin after being passaged in 'nude' mice over 1 year. Chromatography of extracts and culture media of a bronchical carcinoma demonstrated that, in contrast with the other tumours, it secreted a form or forms of calcitonin having size, charge and immunological differences when compared to calcitonin M. Preliminary evaluation of plasma immunoreactive calcitonin estimations in patients with breast carcinoma showed that twenty-three out of twenty-eight patients with metastatic disease had elevated plasma calcitonin concentrations, whereas only one out of thirteen with localized disease had high levels.

Present methods and systems for the generation of human monoclonal antibodies are briefly reviewed. The specificities of the available reagents are outlined. It would appear that the generation using hybridoma methods of human monoclonal antibodies to human tumor cell surface antigens is a rare event and that methods of in vitro immunostimulation may have to be used if such antibodies are to be reliably produced.

A reliable immunoperoxidase schedule for the cellular demonstration of tumour and normal cell products which can be completed in well under two hours is described. The preparations are as clear and readable as those stained by routine histological techniques and should form a valuable adjunct in research and diagnostic studies.

Immune-suppressed mice carrying xenografts of several different types of human germ-cell tumours were injected with a radiolabelled monoclonal antibody (LICR LON/HT13) raised against membrane components of a human germ-cell tumour (HX39). Subsequent assessment of radioactivity in excised organs and tumours showed a selective accretion of antibody in the tumour. Quantitative autoradiography supported the results of radiolocalization observed in vivo in different tumours, and also showed that the antibody localized to viable tumour cells and in close association with their cell membrane. The vascular architecture of tumours was found to be an important factor governing antibody distribution. No localization occurred with radiolabelled normal mouse IgG.

The structural and functional characteristics of nine functioning adrenocortical tumors (four adenomas and five carcinomas) causing Cushing's syndrome or virilization were studied. All tumors that we considered to show histologic evidence of malignant disease and that subsequently metastasized or recurred also showed in cell culture at least one significant functional or behavioral difference from benign tumors. No single defect was common to all carcinomas, but predominant changes included secretion of precursor steroids, such as 11-deoxycortisol (S) and a blunted or absent response to ACTH. All adenomase examined were normal in these respects in comparison with nondiseased cortical cells in culture. In carcinomas whose functions deviated only minimally from normal the presence of highly differentiated ultrastructural characteristics did not, however, confer a better prognosis.

The metabolism of benz(a)anthracene (BA), 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP) by human mammary epithelial cell aggregates in culture has been investigated using non-neoplastic tissues obtained from eight patients undergoing reduction mammoplasty. All three hydrocarbons were metabolized to water-soluble and organic solvent-soluble products and the latter included both K-region and non-K-region dihydrodiols. The major dihydrodiols detected as metabolites of the parent hydrocarbons were the 8,9-dihydrodiols of BA and DMBA and the 9,10-dihydrodiol of BP. The 1,2-dihydrodiols of BA and DMBA and the 11,12-dihydrodiol of BP were not detected. The hydrocarbons also became bound to the proteins and DNA of the epithelial cells but there were wide differences in the extents of binding occurring with the different hydrocarbons and in the extents of metabolism and binding occurring with tissue preparations from different patients. Some of the hydrocarbon-deoxyribonucleoside adducts formed from DMBA and BP appeared to have arisen through reactions of "bay-region" diol-epoxides with DNA, but only very low levels of reaction with DNA were detected in tissue preparations treated with BA.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.

A dual radionuclide subtraction technique for external detection of tumours has been evaluated to determine the viability of the method for use with radioisotope labelled antibodies. A number of external scintigraphic investigations have been carried out with 131I-labelled antibodies to carcinoembryonic antigen (CEA). The investigations were performed on patients with metastatic disease known to produce CEA. The dual radionuclide subtraction technique was used to account for the blood and tissue background. The 131I-labelled antibodies were found to localise in the metastatic lesions, but the subtraction technique using 99Tcm-labelled HSA and pertechnetate gave ambiguous results, which included the production of artefacts. The ambiguities noted in the clinical results were substantiated by experimental data, which highlight the unreliability of this technique.

Current methods employing tumour markers to detect small amounts of residual disease are reviewed with respect to human germ cell, colorectal and breast neoplasms. It is concluded that the assay of tumour markers in body fluids has only limited clinical value. Alternative approaches, such as are afforded by radioimmunodetection and immunocytochemistry, are presented and critically appraised.

An autoradiographic technique is reported for the in vivo localization in the Syrian hamster of small doses of the opiate antagonist, 3H-diprenorphine. The regional distribution in the hamster brain was similar to that reported in the rat. The adrenal medulla showed a high degree of localization providing supportive evidence for the presence of opiate receptors in the medulla and a possible paracrine function for opioid peptides in this site. Other peripheral sites showed selective uptake of 3H-diprenorphine which was not apparently related to the vascularity of the tissue.

The alpha1 and alpha2-globulins have been studied at various stages in the evolution of colo-rectal cancer. The alph2 was elevated in some primary tumours and rose in metastatic cancer especially when it involved the liver. Some apparently tumour free patients had an unexplained elevation of alph2-globulins. The macroglobulins were not a major constituent of the raised alpha2-globulins. Haptoglobulin levels were found to be a useful indicator of tumour activity, when their level was raised in metastatic cancer it was usually with an antecedent or coincidental rise of plasma CEA. Primary tumours may cause a high haptoglobin response without an elevation of CEA.

Forty-one postmenopausal patients with advanced breast cancer have been treated with trilostane, a 3 beta D-hydrogenase delta 5-isomerase inhibitor, for periods of up to 1 year. One patient responded to trilostane and in six patients the disease was stabilized. The remaining patients failed to respond to therapy. Six patients who failed to respond to trilostane subsequently responded to other forms of endocrine therapy. It is concluded that trilostane alone is not a useful agent in the treatment of advanced breast cancer.

42 patients with metastatic breast carcinoma were treated with aminoglutethimide, which inhibits adrenal steroid hormone synthesis. Treatment was stopped in 2 patients before response could be assessed; of the other 40, 15 (37.5%) had an objective response, 1 (2.5%) showed a response in bone but not in soft tissue, and 4 (10%) had complete or very great relief of metastatic bone pain but no radiological evidence of improvement. 19 (53%) of 36 patients with bone metastases responded to treatment (15 had X-ray evidence and 4 had pain relief), as did 5 (45%) of 11 patients with soft tissue metastases, 2 (25%) of 8 with malignant marrow infiltration, 1 (14%) of 7 with lung metastases, and none of 13 with liver metastases. Response was commonest in patients who had previously responded to other forms of endocrine therapy. Side-effects, usually mild and transient, occurred in a few patients; the most important were an initial period of somnolence in 9 patients and a rash in 5.

Ten tumor markers were measured in serum or urine at approximately three month intervals in patients with breast cancer following mastectomy but before development of overt metastatic disease. In 23 patients who later had metastases, only three markers, alkaline phosphatase, carcinoembryonic antigen (CEA), and gamma-glutamyl transpeptidase (gamma-GT) were consistently abnormal prior to the development of detectable metastases in more than one patient. In half the patients, a "lead interval" of three months or more was obtained using these three markers and little advantage was obtained by the addition of any other biochemical marker. The value of these three measurements was then assessed in a larger group of patients and compared with other tests for metastases. Alkaline phosphatase, CEA, gamma-GT, clinical examination, and chest x-ray were the best indices of the metastatic state in breast cancer, being collectively abnormal in 98% of patients at first presentation with metastases. The authors recommend screening patients postoperatively with these five tests for metastases; more detailed tests should only be carried out if results of one or more these are abnormal.

Protein moieties of various molecular sizes and possessing 5-ene-3beta-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118 000, with an isoelectric point between 7-2 and 7-4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17alpha-hydroxy-pregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrated specificity.

A hypernephroma removed from a male patient who had lost 30 kg in weight in the two months preceding surgery was grown as a non-metastasizing transplantable xenograft in immune-suppressed mice. The tumour produced a considerable weight loss (greater than 25 per cent) in the mice at a stage when it comprised less than 5 per cent of the total body weight. A slight fall in food intake of the tumour-bearing mice was noted, but animals bearing other non-cachectic mouse and human tumours had much lower food intakes without accompanying weight loss. No obvious defects in gastrointestinal absorption were detected nor was any gross increase in basal metabolic rate observed. The precise mechanism producing the severe cachexia remains to be established, but elaboration of humoral factors by the tumour seems probable. This model of cachexia bears a closer relation to the clinical situation than do other experimental animal tumour models currently available.

Dendritic cells which express Ia antigen have been demonstrated for the first time in the lactating rat mammary gland. Ultrastructurally, the dendritic cells appear as electron-lucent pale cells interspersed among the epithelial cells of the alveoli, forming a cell population distinct from classical macrophages. They show morphological resemblance to the dendritic cells of lymphoid organs as well as the Langerhans cells of skin. The Ia antigen has been localised by electron microscopic immunocytochemistry on the cell membrane and endocytotic vesicles and tubules. Ia positive cells are also seen in the stroma of the mammary gland. It is proposed that the dendritic cells of the mammary gland belong to the lineage of epidermal Langerhans cells and lymphoid dendritic cells, subserving an immunological role in the lactating breast.

Metastatic breast cancer cells were found in the bone marrow of 60 (23%) of 269 patients with primary breast cancer, none of whom had metastatic disease disclosed by any other investigation, including bone scanning and radiological skeletal survey. We estimated the number of cancer cells as less than or more than 20 cancer cells seen. Twenty-six patients had less than 20 cancer cells present, and 34 had 20 or more. At a median follow-up time of 22 months, 53 patients had relapsed, 19 of 60 (31.7%) in the group found to have micrometastases and 34 of 195 (17.2%) in the group that had normal bone marrow. Patients with micrometastases are relapsing at a faster rate than those without micrometastases (P = less than 0.05). Patients with less than 20 cancer cells present are relapsing faster than those with no cancer cells but slower than those with 20 or more cancer cells (P = less than 0.01). We conclude that the presence of cancer cells in the marrow at primary diagnosis is a prognostic factor in patients with primary breast cancer.

The presence of a tumour-associated immune response in 37 patients with ovarian cancer as assessed by blastogenesis (lymphocyte transformation) evoked by ovarian cancer cell extracts, has been correlated with survival following the test. The difference in these responses is unlikely to be accounted for on the basis of general impairment of cell-mediated immuno-competence. Serum carcinoembryonic antigen (CEA) was also determined in 27 ovarian cancer patients to assess its prognostic significance. Raised CEA levels and absence of blastogenic response to tumour cell extract during relapse are associated with a worse prognosis but neither of these parameters are significant in remission. Possible applications of these findings to the clinical management of ovarian cancer patients are discussed. Serum alpha feto-protein levels measured by radioimmunoassay were not found to be raised in any of the 32 ovarian cancer patients in whom it was measured.

A hypernephroma removed from a male patient who had lost 30 kg in weight in the 2 months preceding surgery was established in immunosuppressed CBA/Lac mice as a nonmetastasizing transplantable xenograft. The xenografted tumors, although comprising less than 5% of the total body weight of the mice, produced considerable weight loss (greater than 25%). A slight reduction in food intake of tumor-bearing mice was noted, but some animals bearing mouse or human tumors not inducing cachexia had equally low food intake without accompanying weight losses. No obvious defects in gastrointestinal histology or absorption were observed. The precise mechanism(s) producing the severe cachexia remains to be established.

The routine preparation of gram quantities of lobuloalveolar and ductal structures from human reduction mammaplasties by treatment with collagenase is described. When cultured on plastic or glass surfaces these structures give rise, initially, to epithelial sheets consisting of cells which retain many morphological characteristics of myoepithelial cells and do not stain with an antiserum which reacts with the surfaces of the lining epithelium of breast ducts and lobulo-alveoli. Subsequently, cells which resemble the lining epithelial cells migrate from these structures and react strongly with the antiserum. Cell proliferation in these cultures is minimal. Explants of major ducts dissected from mastectomies produce vigorously proliferating epithelial sheets which contain morphologically similar cells not identifiable as either lining epithelium or myoepithelium, although nearly all the cells in direct contact with the medium react with the lining epithelium specific antiserum.

The carcinoembryonic antigen (CEA) was produced and released by human colorectal carcinomata and also the normal attached mucosa when maintained in both monolayer and organ culture. Immunoperoxidase cytochemical methods were employed for the cellular localization of CEA which was demonstrable only on the neoplastic cells. Gel filtration and immunological methods confirmed that CEA, produced by normal and neoplastic cells, had properties similar to "authentic" CEA derived from metastatic colorectal carcinomata. In addition, two other CEA cross-reacting macromolecules, neither of which was CCEA-2, were produced by these tumours in culture.

Treatment of mice with 2'-deoxycoformycin (dCf) for 5 days produced inhibition of spleen and lymph node adenosine deaminase (E. C. 3.5.4.4) activity but no hematologic toxicity or weight loss. A 64-fold elevation of erythrocyte dATP was observed. However, if mice were injected with 2'-deoxyadenosine (AdR) in combination with dCf, weight loss, hematologic toxicity, and liver cell necrosis occurred. These mice had a severe blood coagulation defect and a 73-fold elevation of plasma alanine transaminase activity, plasma prealbumin became undetectable, and erythrocyte dATP levels were elevated 1500-fold. Death during treatment appeared to be from acute liver failure since bone marrow toxicity was only detected following termination of treatment. These effects were not seen in mice receiving adenosine in combination with dCf. dCf, either alone or in combination with AdR, inhibited the contact sensitization to oxazalone in mice. The inhibition was associated with signs of systemic toxicity which were more pronounced in the combination-treated groups. If dATP is the toxic metabolic accumulated in the malignant cells of patients treated with dCf, we propose that AdR supplementation of treatment should be considered with extreme caution since severe damage to normal tissues might result.

Monoclonal antibody LICR-LON-M18 is a marker of normal human breast epithelial cell differentiation. The epitope recognized by LICR-LON-M18 is a prominent component of luminal plasma membranes of nonneoplastic resting and lactating human breast epithelial cells but is rarely expressed by human breast carcinomas. With the use of competitive binding-inhibition studies, the immunodominant portion of the LICR-LON-M18 epitope was shown to be the following oligosaccharide sequence [with galactose (Gal) and N-acetylglucosamine (GlcNAc)]: Gal beta 1----4GlcNAc beta 1----. This structure was distinct from Gal beta 1----3GalNac, which was bound by peanut agglutinin (PNA) and was not recognized by LICR-LON-M18 [corrected]. With the use of biochemical techniques, the present data not only confirmed sialylation and consequent "masking" of the LICR-LON-M18 epitope and PNA determinants in human breast carcinomas but also identified the particular groups of glycoproteins involved in this process. These studies provided additional support for the thesis that sialylation of human breast carcinoma glycoproteins represented an enhancement of specific differentiation events normally regulated in the morphogenesis of nonneoplastic human breast epithelium and that specific glycoproteins became masked during the genesis of primary human breast cancer.