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A simple method was developed to isolate low abundance hormone receptor poly(A)+ RNA from cells in tissue culture. Adherent cells in tissue culture plates were directly released with proteinase K and solubilized in SDS. Oligo(dT) cellulose was directly added to the lysate to obtain poly(A)+ RNA. Yields and purity of the poly(A)+ RNA were comparable to other more lengthy methods. IGF-I receptor and insulin receptor mRNA could be detected on Northern blot without any degradation.

There are conflicting reports of seasonal changes in steroid hormone receptor levels in breast cancer tissue. Estrogen receptor and progesterone (PR) receptor levels from 1132 tumors were thus grouped according to month of initial tumor detection or month of tissue sampling/surgery. There was a significant circannual variation in the mean monthly PR receptor concentration in patients grouped according to month of tissue sampling/surgery with peak PR levels in April (late summer-early autumn) and nadir values in August and September (late winter-early spring). There was no significant cyclic variation in estrogen receptor values. A significant annual variation in tumor PR concentration was also seen when receptor levels from individual tumors were grouped according to month of initial tumor detection, with peak PR levels found in January and February. The time interval between tumor detection and biopsy/surgery was 3.3 +/- 5.3 months (mean +/- SD) which was close to the interval between the peak PR concentration expressed by month of tumor detection compared with month of tissue sampling for receptor assay. There was also a significant seasonal variation in the month of initial tumor detection, with peak detection occurring in December (summer). The close synchrony between month of maximum tumor detection and month of peak PR concentration suggests that seasonal changes in detection of breast cancer may in part relate to seasonal changes in hormone responsiveness within tumor tissue.

Tumors of five groups of patients, with (1) nonpalpable primary breast cancer, (2) palpable operable primary breast cancer, (3) loco-regionally advanced primary breast cancer, (4) first and (5) late metastatic breast cancer, were studied in respect to their steroid receptor content. A statistically significant decrease of progesterone receptor positive tumors and of tumors positive for estradiol and progesterone receptors, was found with increasing advance of the disease. A reversed extrapolation of these figures supports the hypothesis that every breast cancer contains steroid receptors and is hormone-dependent from its inception.

Estramustine phosphate, an anti-prostatic cancer agent, was investigated on eleven patients to evaluate the efficacy in a treatment of advanced breast cancers. The daily dose of medication was 840 mg. According to criteria of Japan Society for Cancer Therapy, none was assessed as CR, three as PR, four as NC and PD. The response rate was 27.3%. There was no differences in response rates among estrogen receptor status. A favourable response was observed in postmenopausal patients but no response in premenopausal, as well as a good response in lesions of soft tissue and lung, a poor response in lesions of liver and bone. As to toxicity of estramustine phosphate, gastrointestinal disorders such as nausea, vomiting and diarrhea were noted frequently during the treatment, and a long term administration was not able to perform in premenopausal patients because of vaginal bleeding and discharge, and pain in breast. The estramustine phosphate therapy for advanced breast cancers was regarded as one of modalities for a treatment of postmenopausal patients as a second line therapy. This is the first report in Japan discussing the efficacy of estramustine phosphate for a treatment of breast cancer.

We have synthesized three peptides with amino acid sequences corresponding to amino acids 533-547, 597-611, and 765-779 of the human progesterone receptor (hPR). These peptides were conjugated to keyhole limpet hemocyanin and injected into mice and rabbits to develop antibodies to hPR. Antibodies to the undenatured form of PR were elicited only by the peptide with amino acid sequence 533-547. Fusion of SP2/0 myeloma cells with spleen cells from mice immunized with this peptide produced several active clones. Rabbit sera from immunized animals produced one antiserum that reacted with the undenatured form of PR. One monoclonal antibody (PR-AT 4.14) and one antiserum (PR-AT533) raised against peptide-(533-547) were characterized. Binding of these antibodies to the undenatured form of PR was demonstrated by analysis of the antibody-receptor complexes on sucrose density gradients and by immunoprecipitation techniques. Binding of PR to the antibodies was inhibited by excess peptide. The antibodies did not react with estrogen, glucocorticoid, or androgen receptors, but recognized PR from human breast cancer as well as calf, rabbit, mouse, and rat uteri, indicating that this epitope was conserved among these species. Based on sucrose density gradient analysis of PR prepared and labeled in the presence of proteolysis inhibitors and sodium molybdate, the antibodies bound to a site on the intact undenatured PR, but failed to bind to partially degraded steroid-binding form of the receptor, suggesting that the antibody-binding domain is at or near a site sensitive to proteolysis.

Serum levels of tissue polypeptide antigen (TPA) are related to the proliferative activity and to the mass of the malignancy, differently from any other available tumor marker. We therefore evaluated TPA in comparison with CA15.3 and MCA (mucinous-like carcinoma-associated antigen) in patients with primary breast cancer. TPA was measured in tumor cytosol and in serum. Cytosol and serum TPA levels were not significantly correlated. Serum TPA was higher in patients with locally more advanced disease and in receptor-negative cases. The relation between TPA and disease spread was not directly dependent on tumor bulk, whereas CA15.3 and MCA were highly correlated to the number of positive lymph nodes and tumor size. No correlations were found between TPA and CA15.3 or MCA, and the positivity concordance rate between TPA and CA15.3 or MCA was very low. Patients with higher TPA serum levels showed a worse prognosis in cases with and in those without axillary metastases. From our data we conclude that TPA provides information different from that obtained with breast-specific tumor markers and could therefore be useful in association with CA15.3 and/or MCA in the management of patients with breast cancer.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

Several immunohistological methods for the demonstration of progesterone receptors were tried on routinely processed paraffin wax sections of breast carcinoma, using Abbott's PgR-ICA monoclonal antibody. The best results were obtained with the avidin-biotin-immunoperoxidase complex method with no prior trypsinisation or DNAse digestion, and with imidazole added to the final diaminobenzidine developing solution. A simple semiquantitative scoring system was used to assess the staining results which were then compared with the results obtained by a standard dextran-coated charcoal biochemical assay. Of 31 cases examined, the results of the two methods were concordant in 25 (81%) of cases. This is near the higher end of the concordance range obtained by several other authors using frozen sections. The discordance encountered in a few cases was possibly the result of sampling errors which are more likely to occur with the chemical rather than the histological method. It is concluded that the method described here is fairly reliable and would greatly simplify the process of assessment of progesterone receptors in breast, and possibly other tumours.

We reported the successful treatment of disseminated breast cancer with medroxyprogesterone acetate (MPA). The patient was a 52-year-old female with brain and bone metastasis developed 1 year after surgery. The primary tumor was ER-positive, and she had been treated previously with adjuvant therapy consisting of UFT and tamoxifen. Brain metastasis was surgically removed, but the following combination chemotherapy (epirubicin and vindesine) failed to result in further improvement. Then MPA (1,200 mg/day) was administered as the second-line therapy. After 6 months, multiple bone lesions showed remarkable calcification (PR) and disappeared completely (CR) 13 months later. But the patient was forced to discontinue MPA because of uncontrollable hyperglycemia. At this writing, CR was still being continued and the patient was enjoying favorable quality of life without any treatment. We confirmed that MPA was effective as the second-line treatment for disseminated breast cancer.

HER-2/neu protooncogene amplification and protein expression were analyzed with slot blot and Western blot techniques, respectively, in more than 300 invasive primary breast tumors of all stages. Amplification (2- greater than 30 copies) was found in 17% of these tumors and high expression was seen in 19%. There was a striking coincidence between gene amplification and high expression. Tumors associated with many involved axillary lymph nodes or with Stage IV disease were more often HER-2/neu amplified or overexpressed. Furthermore, gene alteration was strongly correlated with the absence of steroid receptors and with larger tumor size. High expression without gene amplification was seen in a minor subset of tumors of less aggressive character. Neither amplification nor overexpression was correlated with disease outcome for patients with negative axillary lymph nodes. For node-positive patients, however, HER-2/neu amplification was a significant predictor of early relapse and death (median follow-up = 45 months), and a similar trend, although not significant, existed for high gene expression. Multivariate analyses indicated that HER-2/neu alterations were not independent predictors of patient outcome.

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.

The purpose of this study was to investigate the biological behavior of male breast cancer. We evaluated 11 cases of male breast cancer with respect to tumor growth, extent of disease, hormone receptor status, and histological grade of the malignancy, in comparison with 241 cases of female breast cancer. The duration of symptoms was 8.6 +/- 9.1 months in males and 8.5 +/- 18.6 months in females. The incidences of stages I, II, and III were 46%, 27%, and 27%, respectively, in male breast cancer, and 38%, 49%, and 13% in female breast cancer. Metastasis to the lymph node was negative in 60% of the male patients and 54% of the female patients. All cases of male breast cancer were histologically grade I according to Bloom's classification; the histological grades were as follows for the female breast cancer cases: grade I in 99 patients, grade II in 87, and grade III in 55. The rates of hormone receptor positively were 89% for ER and 86% for PgR in male breast cancer, and 64% for ER and 44% for PgR in female breast cancer. Therefore, there was no significant difference in the growth of male breast cancer and female breast cancer, but in male breast cancer the rate of hormone receptor positivity was high, endocrine therapy was effective, and the histological grade was low. Accordingly, the result following appropriate treatment of male breast cancer should be at least comparable to the results with female breast cancer.

A number of investigators have suggested treatment of precursor lesions for invasive breast cancer such as ductal carcinoma in situ with antiestrogen. However, very little information is available on the incidence of estrogen receptor in such lesions and the probability of treatment success. Fourteen formalin-fixed tissue specimens of intraductal carcinoma in situ from 14 female patients aged 40 to 66 years were evaluated for the presence of estrogen receptor by immunoperoxidase technique using estrogen receptor antibody. Eight of the 14 lesions (57%) were positive for estrogen receptor. The incidence of estrogen receptor in intraductal carcinoma in situ is very similar to that of invasive carcinoma of breast, leading to the speculation that ER-positive invasive carcinoma originates from ER-positive precursor lesions. Since only 70 per cent of positive receptor lesions are expected to respond to antiestrogens, it appears that only 40 per cent of patients with ductal carcinoma in situ of breast will benefit from endocrine therapy.

Application of systemic adjuvant therapy for primary breast cancer patients requires a more accurate identification of patients at high risk for recurrence. We have quantitatively assessed the cytosolic levels of estrogen-regulated pS2 protein in tumors of 205 breast cancer patients (median follow-up, 47 mo). There were no significant associations between the level of pS2 protein and tumor size, lymph node status, and differentiation grade. Using length of relapse-free survival (RFS) and overall survival (OS) as end points, 11 ng of pS2 protein/mg of cytosol protein were found as the best cutoff level to discriminate between positive (pS2+) and negative (pS2-). Patients with pS2- tumors showed significantly shorter RFS and OS (P less than 0.0001) than patients with pS2+ tumors. Also after adjustment for tumor size, lymph node status, and estrogen receptor (ER) status, pS2 negativity was associated with earlier recurrence and death. Tumors positive for pS2 (55 of 205, 27%) were almost exclusively confined to the subclass of ER+ tumors (53 of 55, 96%). The death rate for patients with pS2+ tumors was one-tenth of the death rate for patients with pS2-/ER- tumors. In the patients with ER+ tumors, the prognostic power of the pS2 status was especially present in patients whose tumors were also positive for the progesterone receptor (5-yr RFS and OS, 85% and 97% for ER+/PgR+/pS2+ tumors compared with 50% and 54% for the patients with ER+/PgR+/pS2- tumors). In patients with axillary lymph node involvement (N+), pS2 status could discriminate strongly between a good and bad prognosis group (5-yr RFS and OS, 65% and 88% for N+/pS2+ compared with 32% and 34% for N+/pS2-). A similar phenomenon was observed in patients without axillary lymph node involvement (5-yr RFS and OS, 89% and 95% for N0/pS2+ compared with 58% and 82% for N0/pS2-). We conclude that the pS2 status of human primary breast tumors is an important variable for the identification of patients at high risk for recurrence and death. Knowledge of the cytosolic pS2 status appeared of particular importance to identify patients at high risk in the ER+/PgR+ subclass of tumors, and in both the N0 and N+ subclasses of patients.

Frozen sections of breast tumor tissue have been stained using an immunoperoxidase [estrogen receptor (ER)-immunocytochemistry] kit incorporating a monoclonal antiserum [H222] to visualize nuclear human ERs. Quantitation of specific staining has been performed by manual procedures using optical microscopy and by a computer-assisted image analysis system (CAS 100). Initial investigations with a test panel of ER-immunocytochemistry-positive tumors revealed a good qualitative agreement between CAS and manual assessments. Reduced variance was, however, observed between quantified ER-immunocytochemistry results from four experienced investigators using the CAS analysis. An extended study confirmed the relationships between CAS and manual methods of assessment. These findings were evident when studies were scored either by assessment of the percentage of positively stained cells (n = 92; r = 0.919; P less than 0.01) or by H-score calculations (n = 92; r = 0.913; P less than 0.01). A good correlation was also found between CAS quantification and the results of an ER enzyme immunoassay of 48 primary breast cancer specimens (r = 0.715; P less than 0.05). In 49 cases it was possible to relate CAS-defined ER status and levels to the subsequent response of patients to endocrine therapy. ER was assessed on specimens obtained prior to commencement of treatments for recurrent breast cancer. Presuming the presence of ER to be a prerequisite for successful therapy, very good correlations between response and both status and levels of positivity were recorded. None of 16 patients with CAS-ER-negative tumors responded to treatment, while 16 of 33 (48.4%) CAS-ER-positive patients achieved an objective response according to International Union Against Cancer criteria. A relationship between response and the degree of CAS-ER positivity was obtained when the CAS score divisions of 0, 1-100, and greater than 100 (response rates, 0, 41, and 64%, respectively) were used. These data demonstrate that automated image analysis offers a reliable, reproducible procedure for quantifying ER in immunocytochemically stained sections. It has potential advantages over manual procedures, providing less opportunity for subjective influences in scoring sections. Future advances in software design should further reduce elements of subjectivity and increase both the speed and reliability of results. We anticipate image analysis becoming a valuable tool in investigations concerning, for example, the influence of heterogeneity of steroid receptor distribution on the rate of recurrence of breast cancer after mastectomy and in the clinical course of the disease.

Previous investigations have suggested that high-dose methotrexate with leucovorin rescue is a potentially useful strategy for overcoming antifolate resistance. Interactions between methotrexate (MTX) and leucovorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reductase (MTX/MTX polyglutamates versus dihydrofolate) and other folate-dependent enzymes (MTX polyglutamates versus reduced folate substrates). The present studies were designed to test the ability of dihydrofolate to compete with methotrexate and methotrexate polyglutamates for dihydrofolate reductase activity using an intact human breast carcinoma cell line (MCF-7) as the model system. Exposure of the breast cells to methotrexate for 24 h resulted in a concentration-dependent formation of methotrexate polyglutamates that markedly exceeded the dihydrofolate reductase-binding capacity for up to 24 h after the removal of drug from the growth media. Under these conditions of dihydrofolate reductase inhibition, we found that tritium-labeled dihydrofolate was capable of competing with methotrexate and its metabolites for dihydrofolate reductase activity as evidenced by the appearance of tritium-labeled reduced folates in the treated cells. We found the interaction between dihydrofolate and methotrexate to be dependent on the exposure concentrations of both methotrexate and dihydrofolate. These studies provide direct evidence that competition during leucovorin rescue occurs at the level of dihydrofolate reductase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.

We have earlier described a monoclonal antibody (323/A3) against a Mr 43,000 surface glycoprotein of MCF-7 human breast cancer cells which shows considerable specificity for primary and metastatic breast tumors (Cancer Res., 46: 1306-1317, 1986). Here we report the occurrence of the 323/A3 antigen in a large cohort of primary breast tumors (m = 384) and its interrelationship with several clinically important variables. Frozen, stored tumor tissues were examined by a Western blot procedure, and the level of 323/A3 protein in individual tumors was calculated in arbitrary units based on the integrated Mr 43,000 signal in tumors compared with an MCF-7 internal standard. Thirty-six % (139 of 384) of tumors were found to be positive for 323/A3. Higher frequencies of 323/A3 protein were found in tumors larger than 2 cm (P = 0.03), tumors with infiltrated lymph nodes (P = 0.01), and tumors without estrogen receptor (P = 0.006). No significant relationship was found with patient age, menopausal status, or progesterone receptor status. Of the newer clinical determinants proliferative rate (% S phase), DNA ploidy, and the lysosomal protease cathepsin D, but not the HER-2/neu oncogene protein, were significantly correlated with 323/A3. The presence of 323/A3 protein was also related to increased recurrence (P = 0.003) and mortality (P = 0.036) after primary treatment. As an exposed surface antigen, this glycoprotein might be a useful target in radioimaging and immunotherapy of some human breast tumors, especially those having large size, infiltrated lymph nodes, deficient estrogen receptor, high proliferative rate, abnormal DNA content, and high levels of cathepsin D, all of which are ominous indicators of tumor behavior.

Size of tumor has not been established as a predictor of tumor recurrence in the breast following conservative surgery and radiation therapy. We analyzed 783 patients with infiltrating carcinoma treated with simple excision and radiation therapy. Median follow-up was 91 months. Median age at diagnosis was 50 years. There was a 13% recurrence among patients with T1 lesions compared with a 12% recurrence among patients with T2 tumors. Size did not predict for local recurrence when the tumor was analyzed by 1-cm increments and whether the tumor was estrogen receptor protein positive or estrogen receptor protein negative. Patients with an extensive intraductal component had a significantly higher local recurrence rate for every tumor size compared with patients with extensive intraductal component-negative tumors. We concluded that size did not predict local recurrence.

To clarify the accuracy of immunocytochemical detection of estrogen receptors (ER) in breast carcinomas using cytological materials, imprint specimens from tumor tissue were compared with frozen tissue sections and tumors analyzed by the dextran coated charcoal (DCC) method and enzyme immunoassay (EIA). Out of 50 cases examined by imprint immunocytochemistry, there were 39 ER positive cases (78.0% positivity). The positivity in the imprint materials agreed with that of the DCC in 36 out of 40 cases (85.0%), with 100% sensitivity and 60.0% specificity. The two methods statistically correlated with each other in their positivity and grade (p less than 0.001). The positivity and grades of imprint and frozen immunocytochemistry as well as those of imprint immunocytochemistry and the EIA agreed almost perfectly with each other. As a result of the present study, we concluded that immunocytochemical detection of ER is indeed reliable, as accurate as other procedures. We recommend that aspiration biopsy cytology (ABC) be used for morphological examination and ER immunocytochemistry when adequate materials are available and that imprint materials be used when ABC materials are inadequate and fresh tissue is available at the time of surgery.

Forty-nine patients with locally advanced breast carcinoma were prospectively randomized to be treated with either CMF or CMF plus T for four courses, both before and after mastectomy. The overall clinical objective remission rate for induction treatment was similar (71% with CMF and 64% with CMF + T). The time to progression or recurrence of the disease was also not significantly different between the two groups. Overall survival was shorter after CMF + T treatment (median value of 41.5 months) than after CMF treatment alone (median value of 79.7 months; p = 0.05). Even after progression or recurrence, survival was shorter for patients receiving CMF + T than those receiving CMF (median values of 7.5 and 17.3 months, respectively; p = 0.09). These results show that the addition of T to CMF in the treatment of locally advanced breast carcinoma, before and after mastectomy, offers no advantage for improving the overall response rate. Moreover, this addition may have an adverse impact on survival in this disease setting.

To determine whether we could replace our current binding assay (BA) method for measurement of estrogen receptors (ERs) and progesterone receptors (PRs) with the recently developed enzyme immunoassay (EIA) method, we compared simultaneous measurements of ERs and PRs in frozen breast tumor samples by both methods. A value of greater than or equal to 10 fmol/mg cytosol protein was defined as positive. There was agreement between the BA and EIA on whether the sample was positive or negative in 75 of 91 (82%) samples measured for ERs and in 74 of 93 (80%) for PRs. When the threshold value for a positive assay was redefined as greater than or equal to 20 fmol/mg protein, there was agreement in 85 of 91 (93%) samples for ERs and 85 of 93 (91%) for PRs. The numerical value for ERs by EIA was not consistently greater or less than ERs by BA, but the difference between the EIA and BA measurement increased as the size of the measurement increased. We did not see an excess of premenopausal patients whose ERs by BA were negative and whose ERs by EIA were positive. Although we performed a linear regression analysis and determined the Pearson correlation coefficient to compare the BA and EIA as reported by others, we show that this analysis may be misleading when the objective is to demonstrate similarities between these methods. Our study shows that the EIA can be confidently used in place of the BA. However, a threshold value for a positive EIA should be confirmed clinically in future studies.

The effect of systemic therapy on tumour oestrogen receptor (ER) concentration has been studied in 88 patients with large, operable, primary tumours (total 89) of the breast. In 26 patients, tumour was not available for study on one occasion (usually post-treatment). Forty-five patients were treated initially by endocrine therapy but, of these, 13 who had failed to respond went on to receive chemotherapy also. Seventeen patients with low concentrations of ER (less than 20 fmol mg-1 protein) were treated directly by chemotherapy. Patients underwent an incisional biopsy for confirmation of diagnosis and determination of pre-treatment ER by radioligand binding assay, followed by systemic therapy for 3 months (or 6 months for both endocrine and cytotoxic therapies). Response was assessed clinically and mammographically before mastectomy. ER concentration was then determined in the post-treatment tumour specimen. No significant change in ER concentration was seen in any treatment group except when the patients had received tamoxifen; there, receptor concentration fell to very low levels, presumably due to interference with the assay. There was no relationship between tumour response to systemic treatment and change in ER concentration. It is concluded that changes in ER concentration are unlikely to play a major role in the early response of breast tumours to systemic therapy.

The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. We have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembraneous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. In such cultures the boundary between apical and basal domains was observed at the point of cell contact with the substratum. Immunocytochemical analysis of these cell-substratum contacts revealed the absence of a characteristic basement membrane containing laminin, collagen (IV), and heparan sulfate proteoglycan. However, serum-derived vitronectin was associated with the basal cell surface and the cells were shown to express the vitronectin receptor on their basolateral membranes. Additionally, treatment of cultures with antibodies against the vitronectin receptor caused cell detachment. We suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS-O while colchicine and acrylamide did not. We hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

The expression of oestrogen regulated protein, P24, was investigated in 69 breast cancers. At initial evaluation P24 protein was detected significantly more frequently and was present in significantly higher concentration in oestrogen receptor positive than in receptor negative tumours. There was, however, no correlation between P24 staining and progesterone receptor, tumour ploidy or proliferative index. Nineteen patients received a short course of treatment with diethylstilboestrol. Following treatment with oestrogen, P24 staining became positive in 7/13 tumours previously negative for P24, including six tumours which were oestrogen receptor negative. Oestrogen administration also caused an increase of the proliferation index in 12/19 tumours, including 5/7 that were oestrogen receptor positive and 7/12 that were oestrogen receptor negative. In some instances oestrogenic stimulation of proliferation occurred together with increased P24 expression; in other instances proliferation index increased without induction of P24 synthesis. The in vivo effects of oestrogen in clinical breast cancer thus appear to show dissociation between enhancement of protein synthesis and cellular proliferation.

Four courses of ifosfamide with mesna given by intravenous infusions were combined with bolus doxorubicin in a phase II trial to treat patients with breast cancer. Ifosfamide can deplete intracellular glutathione levels and because doxorubicin resistance may be associated with elevated levels of intracellular glutathione these drugs were combined in an attempt to overcome clinical cytotoxic drug resistance. There were 31 women with poor prognosis advanced breast cancer in the study. Forty-five percent were younger than 40 years old, 68% had visceral dominant disease, 59% had more than two disease sites, and each of the five women tested had increased expression of primary tumor epidermal growth factor receptor. The objective response rate was 71% with manageable toxicity (95% confidence interval, 54% to 85%). The response rate in 22 patients not given prior chemotherapy was 72% and in 9 patients with cancers resistant to previous mitoxantrone monotherapy it was 67%. The high antitumor activity of this combination suggests further exploration of its use in breast cancer.

The isolation and characterization of complementary DNAs (cDNAs) which code for an epithelial antigen aberrantly expressed in human breast tumor tissue are described here. The only information regarding the primary structure of this potentially important antigen has been a 20-amino-acid repeat motif. We now report the complete amino acid sequences of different forms of the human epithelial tumor antigen as deduced from the nucleotide sequence of isolated non-repeat cDNAs. The diversity of protein forms is generated by a series of alternative splicing events that occur in the regions located upstream and downstream to a central tandem repeat array. Isolated cDNAs coding for the upstream region show that differential usage of alternative splice acceptor sites may generate two protein forms containing putative signal peptides of varying hydrophobicities. The complexity of possible antigen forms is further compounded by alternative splicing events occurring in the region 3' to the repeat array. The isolated cDNAs 3' to the tandem repeats indicate that whereas one mRNA transcript is colinear with the gene, and defines an open reading frame (ORF) containing 160 amino acids downstream to the repeat array, a second cDNA correlates with a mRNA that is generated by a series of splicing events. The deduced amino acid sequence of the spliced cDNA contains an ORF that is identical for 149 amino acids downstream to the repeat array with the amino acid sequence of the unspliced cDNA. At this point it diverges and continues for an additional 179 amino acids. The sequence contains a highly hydrophobic 28-amino-acid peptide, located towards the carboxyl terminus, that may correspond to a transmembrane region. The cDNAs and deduced amino acid sequences, presented here, define the complete amino acid sequences of the epithelial tumor antigen and demonstrate the existence of multiple protein forms that probably localize to different cellular and extracellular compartments.

We developed a simplified polymerase chain reaction (PCR) technique sensitive enough to detect estrogen receptor (ER) mRNA in breast tumor specimens from 1 microgram of total RNA. We simultaneously amplified a control gene such as beta-actin as a baseline to semiquantitate ER expression. In a preliminary test of this method on a small series of breast tumors, ER message was found as expected in a number of tumors found to be ER positive by ligand binding assay, but also ER negative in one tumor assayed. The ER in this last tumor contained a single base change in the hormone binding region, compared with the MCF-7 breast tumor cell line ER. Therefore, this PCR technique may be useful in the detection and cloning of rare ER transcripts from breast tumor biopsy specimens.

Breast cyst fluid from 35 women was stratified into risk groups based on personal and family history of breast cancer. Mitogenic activity in breast cyst fluid of women at highest risk to develop breast cancer was significantly higher than the activity in the lowest-risk group. There was a direct dose-dependent relationship between mitogenic activity and increased risk of developing breast cancer. Size-exclusion chromatography showed that breast cyst fluid from women at highest risk contained two peaks of growth factor activity: less than 6 kilodaltons (kd), identified as human EGF (epidermal growth factor), and 6 to 18 kd. Moderate-risk group samples demonstrated only the single less than 6 kd peak, whereas the lowest-risk group had insignificant growth-promoting activity. Breast cancer tissue analyzed in a similar manner revealed a predominant 6- to 14-kd peak of mitogenic activity demonstrating the same acid- and heat-stability found in breast cyst fluid.

The presence of immunoreactive adrenocorticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), and somatostatin has been investigated by immunohistochemistry in forty biopsies from breast cancer patients. All of these hypothalamic hormones were found in about 30% of the samples, seen in the cytoplasm or in the nuclei of the tumor cells. Positive immunostaining for the hypothalamic hormones was present in colloid, lobular, and infiltrating ductal carcinomas. There was not a clear relationship between occurrence of staining for the hypothalamic hormones and the histologic grade of tumors or the clinical stage of the disease. Immunoreactive LHRH was more frequently found in breast tumors with estrogen and progesterone receptors. On the other hand, preneoplastic breast lesions expressed mainly somatostatin, while immunoreactivity was absent in normal mammary tissue.

Recently, it was reported that there is a significant elevation of total plasma apolipoprotein(a) level in cancer patients. Because of their structural homology and immunological cross-reactivity, localization of the plasminogen and the apolipoprotein(a) was comparatively studied in cancerous tissues of breast (N = 4) and colon (N = 3) by immunofluorescence. The following results were obtained: 1) Tumor cells isolated or in nodules were strongly stained in all cancerous samples using antiserum against Pg, absorbed or not with Lp(a). 2) Tumor cells were lightly stained in two breast carcinomas, using anti-Lp(a) serum. All other carcinomas were negative. The staining was abolished when anti-Lp(a) serum was absorbed with Pg. 3) Blood vessels were strongly stained using antiserum against Lp(a) even when it was absorbed with Pg. Anti-Pg serum decorated only a few capillaries. These results show that the two proteins have different localizations: Lp(a) is seen exclusively in the vascular system. The component present at the surface of tumor cells is plasminogen (or plasmin) but not Lp(a).

The c-erbB-2 proto-oncogene encodes a 190 k Mr protein representing a putative growth factor receptor with considerable homology to the EGF receptor. Gene amplification and overexpression of the oncogene protein have been demonstrated in a variety of tumours including breast cancer, where it is associated with a poor prognosis. In this study we have produced and characterized a mouse monoclonal antibody, designated NCL-CB11, to the c-erbB-2 protein. NCL-CB11 was generated to a synthetic peptide sequence corresponding to a site of predicted antigenicity near the C-terminus of the internal domain of the protein. NCL-CB11 produces intense membrane-associated immunohistochemical staining in a proportion of human cancer cells. The specificity of the antibody is supported by Western blotting and immunoprecipitation studies. Reactivity with an internal site of the protein is confirmed by the necessity of cell permeabilization for reactivity in fluorescence-activated cell sorter (FACS) analysis. A high degree of correlation between immunohistochemical staining using NCL-CB11, and c-erbB-2 gene amplification has been observed. NCL-CB11 should prove to be a valuable reagent for investigations into the pathological significance of c-erbB-2 protein expression.

Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.

An immunocytochemical assay using a monoclonal antibody specific for estrogen receptor (ER-ICA) was performed on needle aspirates and on histologic sections (mastectomy and biopsy specimens) from 55 patients with breast cancer. A total of 82 ER-ICAs were performed, with matched cytologic and histologic specimens in 27 patients, cytology alone in 15, and histology alone in 13. ER-ICA results were described by a histochemical score (H score) based on intensity-weighted percentages of staining cells. The H scores were compared with results of sucrose density gradient (SDG) analysis of histologic specimens (mastectomy, resection, or biopsy). An H score greater than or equal to 10 and an SDG value greater than or equal to 10 fmol/mg protein were considered positive. The sensitivity of cytologic ER-ICA was 94%, the specificity 100%. The sensitivity of histologic ER-ICA was 67%, the specificity 90%. Correlating cytologic H score with Black's nuclear grade showed that grade 1 (the most anaplastic) carcinomas demonstrated the lowest H scores (mean, 7.3 +/- 29.8), whereas the highest H scores were noted in grade 3 tumors (mean, 150.0 +/- 88.1). Both SDG and ER-ICA showed ER values to be lower in premenopausal than postmenopausal women. There was no correlation between H score and presence of axillary nodal metastases or tumor size. An overall good correlation was demonstrated between immunohistochemical methods and biochemical analysis.

We have synthesized three peptides with amino acid sequences identical to those spanning amino acids 201-215, 231-245, and 247-261 of the human estrogen receptor (hER). These peptides were conjugated to keyhole limpet hemocyanin and used as immunogens to develop monoclonal antibodies (MoAbs) to hER. Antibody responses were only elicited by the peptide with amino acid sequence 247-261. Splenocytes from immunized mice were used for hybridoma production. Of the seven MoAbs that recognized the native (functional) form of the ER, four (MoAbs 16, 33, 114, and 213) recognized the ER with high affinity, as demonstrated by the increased sedimentation coefficient of the antibody-complexed ER in sucrose density gradients. Antibodies 318, 35, and 36 bound to ER with low affinity since they immunoprecipitated ER, but the ER-antibody complex appeared to dissociate on sucrose density gradients. The high-affinity MoAbs appear to be site-specific since the peptide competed effectively for binding of the receptor by the antibody. The fact that they reacted with ER from human breast cancer and calf, rat, and mouse uterine tissues suggests that this epitope of the receptor is conserved in these species. Although the DNA-binding region appears to be conserved among the various steroid receptors, these MoAbs did not recognize the native forms of progesterone, androgen, or glucocorticoid receptors. These MoAbs bound to the KCl-activated 4S ER and heat-transformed 5S ER, suggesting that the antibody-binding site is accessible in the monomeric and dimeric forms of ER. The antibodies did not recognize the untransformed 8S ER in the presence of molybdate and without KCl, suggesting that the antibody-binding site in the oligomeric form of ER is inaccessible. The fact that the antibodies did bind to the unoccupied 4S ER was demonstrated by the data obtained with sucrose density gradient analysis followed by postlabeling of ER with [3H]estradiol. The antibodies bound to ERs with high affinity (KD = 0.4 to 1.8 nM). At a fixed concentration of antibody, ERs ranging from 20 to 1,000 fmol were detectable. These MoAbs did not inhibit nuclear or DNA binding of ER in vitro. This can be attributed to the dissociation of the antibodies from ER when the latter interacts with its acceptor site. These results demonstrate the development of site-specific MoAbs to the native form of the hER using synthetic peptides as immunogens.

17 alpha-Iodo-vinyl oestradiol binds with high affinity to the oestrogen receptor, is metabolically stable and has been shown to accumulate in oestrogen sensitive tissues of rodents. We have synthesised this compound and its 3-acetate, labelled with carrier free 125I and shown the accumulation of both compounds in rat uterus. 17 alpha-Iodo-vinyl oestradiol-3-acetate was prepared labelled with carrier free 131I and administered to twelve patients with suspected breast cancer, approximately 1 h before surgical removal of the primary tumour mass. At the time of surgery a sample of the tumour and a peripheral blood sample were taken. The ratio of radioactivity in the tumour to blood was determined. All tumours accumulated radioactivity and ratios ranged from 1.5:1 to 3.7:1. There was no correlation between the degree of accumulation and either cytosolic oestrogen or progesterone receptor concentration in the tumour. Analysis of blood revealed a single circulating species, 17 alpha-iodo-vinyl oestradiol. The results show that 17 alpha-iodo-vinyl oestradiol is metabolically stable and accumulates in breast tumours though this accumulation is not sufficient to permit imaging.

To verify the relationship of mammographic (Mx) patterns of breast cancer to hormone receptor content (ER, PgR) we studied 129 women (59% in postmenopause; average age 55) in the last 3 years. All women had operable breast cancer and were submitted to mammography before surgery. The tumors were classified, according to Mx characteristics, in 5 classes (Broberg, 1983). ER and PgR contents (cut-off for positivity: 10 fmol/mg prot cyt) were analyzed in all patients by means of DCC method. The percentage of ER+ cases was significantly higher in class I than in all other Mx classes (85% vs 57%; p = 0.02), whereas it was lowest in class IV (51% vs 70%; p = 0.03). The percentage of PgR+ cases was significantly different only in class I with respect to class IV (70% vs 41%; p = 0.002). As for ER and PgR mean tumor content, no statistically significant difference was observed between the 5 classes. When the 2 receptors were simultaneously considered the percentage of ER+ PgR+ cases was higher in class I than in all the extant classes (p = 0.01), and the percentage of ER- PgR- was higher in class IV than in the extant ones (p = 0.02). In selected subgroups of patients, Mx classification of breast cancer can help the physician predict the hormone receptor tumor status with sufficient reliability.

Using archival paraffin wax embedded tumour we have investigated histological grade, DNA ploidy, S phase fraction and proliferative index in 74 patients with symptomatic ductal carcinoma in situ (DCIS) of the breast. Nine patients developed local recurrence, six invasive in character. No patients with the cribriform subtype of DCIS developed local recurrence. The cribriform subtype showed a significantly lower rate of DNA aneuploidy and a lower proliferative index than the other subtypes. Cribriform tumours were almost exclusively well differentiated in contrast with the comedo and solid variants. Our results suggest the cribriform variant is less aggressive than other subtypes of DCIS. This has possible implications for management of these lesions.

Breast cancer cell lines (MCF-7, T47D, BT-20 and STT-11) and fresh cells from malignant effusions of eight breast cancer patients were examined for their in vitro sensitivity to 17 beta-estradiol (E2), tamoxifen and toremifene in a miniaturized, improved nucleic acid precursor incorporation assay (MINI assay). Seven of the eight patients received either tamoxifen or toremifene following a MINI assay and the correlation was examined between in vitro sensitivity and clinical responses to the hormonal agents. In cell lines, E2 stimulated thymidine incorporation by estrogen receptor (ER)-rich cells, MCF-7 and T47D, but not by ER-poor cells, BT-20 and STT-11. Tamoxifen induced both ER-mediated and -unmediated effects in ER-rich cells. The latter effect was also observed in ER-poor cells. Toremifene had less ER-unmediated effect in all of the cells tested than tamoxifen did. The ER-mediated effect of toremifene was weaker than that of tamoxifen in cell lines but was equipotent to tamoxifen in fresh cells. E2 affected thymidine incorporation by cells withdrawn from patients who showed a partial response to the anti-estrogens. No clear correlation was demonstrated between in vitro sensitivity to anti-estrogens of fresh cells and clinical response to these agents. The present results suggest that 1) the MINI assay is a useful system to investigate hormonal effects on breast cancer cell lines; 2) clinical responses to anti-estrogens are not predicted by in vitro response to the agents but might be predicted by the in vitro response to E2; and 3) toremifene has a smaller non-specific effect on breast cancer cells than tamoxifen and is equipotent to tamoxifen in the ER-mediated effect in vitro.

A series of 90 patients with primary operable breast cancer and with up to 36 months of follow up were investigated for expression of epidermal growth factor receptor (EGFR) in their tumours by immunocytochemical staining with the monoclonal antibody EGFR 1. Tumour samples were snap frozen in liquid nitrogen immediately after resection and subsequently stained using a standard indirect immunocytochemical method. Tumour staining was assessed by two observers and scored on a four point scale (0-3). Thirteen (14%) tumours showed positive immunoreactivity. A strong correlation between distinct EGFR expression and short disease free interval was observed. Significant correlations were also shown with oestrogen and progesterone receptor expression and tumour nuclear size. No significant association was found with tumour size, lymph node stage, and histological grade. The association with disease free interval remained significant in multivariate analysis.

The immunohistochemical and DNA profiles of two cases of florid papillomatosis of the nipple (FPN) were compared with the immunohistochemical and DNA profiles of mammary intraductal carcinomas (IC) to assess the relationship between these two proliferative neoplasms. Both examples of FPN were circumscribed papillary tumors in the subareolar breast that showed cytologic atypia, intraductal necrosis, and a distinct myoepithelial cell layer. An antibody to muscle-specific actin (MSA) decorated a continuous myoepithelial layer in one case that was confirmed by electron microscopy. MSA showed patchy, discontinuous staining of apparent myoepithelium in the ICs. Flow cytometric analysis showed that both FPN lesions were diploid, rapidly proliferating lesions with S-phase fractions of 10.9% and 34.4%. One IC was aneuploid, and the five diploid neoplasms showed S-phase fractions ranging from 6.4 to 15.8%. In FPN many epithelial cells stained intensely for S-100 protein, but each IC also showed at least focal expression of S-100 protein. One case of FPN was focally positive for gross cystic disease fluid protein 15 (GCDFP-15), but neither stained for tumor-associated glycoprotein-72 (TAG-72) nor for the product of the c-erbB-2 oncogene. In comparison, three ICs expressed focal GCDFP-15, four stained for TAG-72, and one was positive for the c-erbB-2 oncogene product. These preliminary observations suggest that the tandem proliferation of epithelial and myoepithelial cells and the preservation of a normal structural relationship between the two appears to separate FPN from intraductal carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

An immunohistochemical study of c-erbB-2 expression was carried out on in situ (non-invasive) breast carcinoma, using antibody 21N, raised to the intracytoplasmic domain of the c-erbB-2 oncogene product. Strong membrane staining was observed in 44 out of 74 (59 per cent) cases of ductal carcinoma in situ (DCIS), but none of 48 lobular carcinoma in situ (LCIS) lesions. A detailed comparative morphological evaluation using several different parameters, including histological subtypes, was performed within the DCIS group. The results showed that there was a significant correlation between c-erbB-2 expression and the presence of large cell size, periductal lymphoid cell infiltration, marked nuclear pleomorphism, multinucleation, and a high mitotic rate. Of these, cell size appears to be the most important predictor of c-erbB-2 status, followed by the presence of periductal lymphoid cell infiltration. These results indicate, firstly, that LCIS and DCIS are biologically (as well as histologically) different and, secondly, that a subgroup of DCIS, which is associated with c-erbB-2 over-expression, exists and appears to have distinct histological features. The subgroup of DCIS cases which over-express c-erbB-2 may be a biologically definable category with prognostic importance. These results may therefore have relevance to breast screening programmes, but a larger study incorporating clinical data would be necessary to correlate these findings with clinical outcome.

Psychosocial correlates of hormone receptor status in primary breast cancers were investigated in 93 consecutive patients. Life event, coping style, and psychological adjustment self-report scales were completed. The 75 patients with receptor-positive (oestrogen and/or progesterone) tumours were better adjusted psychologically than the 18 patients with receptor-negative lesions. These findings may explain the relations that have been found between psychosocial variables and survival.

We used the population-based tumor registry of Kaiser Permanente in the United States (Portland, OR) to analyze breast cancer incidence from 1960 to 1985. Overall, incidence rose 45% during this period. The largest increases occurred in women 60 years of age or older (74%) and in those 45-59 (36%). The rate in women aged 20-44 has remained essentially unchanged. Localized and regional disease showed similar increases. Review of medical records revealed that only a small portion of this increase was likely to result from increased screening activities. From the increased availability of receptor assays in a large proportion of cases since the mid-1970s, we observed that incidence of estrogen receptor-negative cancers rose 22%-27% between the mid-1970s and the mid-1980s. In contrast, incidence of estrogen receptor-positive tumors increased an average of 131% in the same period, perhaps implicating hormonal factors in the rising incidence of breast cancer.

Estrogen receptors (ER) were independently analyzed using dextran-coated charcoal assays (ER-DCC) and immunohistochemical assays in frozen (ER-ICA) and paraffin-embedded tissue (ER-PAR) from 130 human breast cancer specimens drawn from postmenopausal high-risk patients registered in the Danish Breast Cancer Cooperative Group. ER was best detected with the ER-DCC assay followed by the ER-ICA (relative sensitivity 87%) and the ER-PAR assays (relative sensitivity 71%). The semiquantified staining features of the immunohistochemical assays were statistically significantly correlated with each other and with ER-DCC. Analysis of disease-free interval (DFI) and overall survival (OS) showed that all assays allowed statistically significant discrimination between a high risk and a low risk group, although the sensitivity differences tended to be reflected as small differences in clinical discriminatory power. The patient groups were then stratified according to adjuvant treatment [radiotherapy (RT) versus radiotherapy and tamoxifen (RT + TAM)]. The survival advantage was tied primarily to the receptor status itself in the steroid-binding assays, but was linked to both the receptor status and the adjuvant treatment in the immunohistochemical assays. Thus, the relative risks in terms of DFI and OS were of the same relative magnitude in the RT and RT + TAM groups for ER-DCC assays using a cut-off level of 10 fmol/mg cytosol protein, while there were large differences in the relative risks between RT and RT + TAM groups for ER-ICA and ER-PAR assays. We conclude that an ER assay in fresh tissue should be given first priority, but if there is no fresh tissue, an ER assay in paraffin-embedded tissue offers a reasonably good alternative as a prognosticator and an equivalent alternative as a predictor of the response to endocrine treatment.

In 12 cases of human mammary carcinoma in which a preneoplastic atypical ductal hyperplasia was also identified, quantitative DNA (QDNA) measurements of thionein-stained samples from both lesions were performed using the Cell Image Analysis 100 system. The QDNA values in the preneoplastic and neoplastic lesions from each case showed concordance (six as euploid and six as aneuploid/hyperdiploid). Such congruence suggests a stable inheritance of the somatic mutation(s) that is involved in carcinogenesis and that affects ploidy. If this relationship between concurrent preneoplasia and neoplasia in the ipsilateral breast is confirmed, it offers the possibilities of (1) identifying individuals at risk for developing neoplasias with defined biologic characteristics and (2) developing therapeutic regimens more appropriate to the risk assessment of each patient. It may be possible to conceive of a rational preventive regimen for cancer of the breast.

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.

Culture of ZR-75-1 human breast cancer cells for 5 days in the absence of oestrogens (phenol red-free medium supplemented with dextran coated charcoal stripped 5% fetal calf serum) resulted in a slowing of growth rate and loss of progesterone receptors. Oestradiol at 10(-9) M markedly stimulated growth and progesterone receptor synthesis over a 5-day period. While medroxyprogesterone acetate (10(-10) to 10(-6) M) inhibited growth of ZR-75-1 cells growing in complete medium, in the short-term absence of oestrogens low concentrations were growth stimulatory. Cells deprived of oestrogens for 5 days retained sensitivity to growth inhibition by 4-hydroxy tamoxifen. ZR-75-1 cells were also adapted to growth in the absence of oestrogens over a 5-month period. These cells (ZR-PR-LT) failed to express binding sites characteristic of the type 1 oestrogen receptor but progesterone receptor expression was at a level normally associated with oestrogen induction. Adapted cells were growth inhibited by oestradiol, 4-hydroxy tamoxifen and medroxyprogesterone acetate, but despite elevated progesterone receptor expression the progestin was only marginally more inhibitory than in the parent line. Our data indicate a poor quantitative relationship between response to progestins in vitro and progesterone receptor concentration and support previous findings that acquisition of an oestrogen independent phenotype does not necessarily result in resistance to anti-oestrogens.