A controlled trial of adjuvant tamoxifen, with or without prednisolone, in post-menopausal women with operable breast cancer.

A randomised clinical trial has been conducted to compare adjuvant tamoxifen, 20 mg daily, with tamoxifen and prednisolone, 7.5 mg daily, in post-menopausal women with operable breast cancer. There were 254 evaluable patients, of whom 128 were given tamoxifen alone and 126 received tamoxifen and prednisolone. After a median follow-up of 48 months there was no significant difference in relapse-free or overall survival of the two groups. Furthermore, with survival slightly favouring tamoxifen, confidence intervals on the hazard ratio established that a difference in favour of tamoxifen plus prednisolone of even 5% at 5 years was very unlikely (P < 0.02). Thus, despite the relatively small number of patients in this trial, the data clearly establish that prednisolone is not of value as an additional adjuvant agent.

Chemotactic response to mucin by Serpulina hyodysenteriae and other porcine spirochetes: potential role in intestinal colonization.

Chemotaxis of porcine spirochetes towards a variety of mucins was measured quantitatively by a capillary method. A chemotaxis buffer consisting of 0.01 M potassium phosphate buffer (pH 7.0) and 0.2 mM L-cysteine hydrochloride was necessary for chemotaxis of spirochetes. The optimum incubation time and incubation temperature were 1 h and 40 degrees C, respectively. The mucin concentration also affected the chemotaxis observed, and a concentration of 1% (wt/vol) was near the optimum. Virulent Serpulina hyodysenteriae strains were chemotactic towards 1% (wt/vol) hog gastric mucin and 1% (wt/vol) porcine colonic mucin but not towards 1% (wt/vol) bovine submaxillary mucin. Virulent S. hyodysenteriae strains were significantly more chemotactic than avirulent strains of S. hyodysenteriae (SA3 and VS1), Serpulina intermedius, and Serpulina innocens. Other spirochetes belonging to the proposed group of spirochetes Anguillina coli were also not chemotactic. Pathogenicity of S. hyodysenteriae strains that cause swine dysentery may, in part, be attributed to their attraction to porcine intestinal mucus.

Fine-wire localization and biopsy of non-palpable breast lesions.

We have undertaken fine-wire localization and biopsy of 130 impalpable breast lesions identified by mammography and considered suspicious of malignancy. Histologically 22 of these lesions were invasive carcinomas and 24 were in situ carcinomas (35 per cent malignant). Twenty-nine per cent of the lesions were identified during the screening of asymptomatic women. In the remainder, the presenting symptoms bore no relation to the eventual histological diagnosis. Clusters of microcalcification were more often malignant than were abnormal soft-tissue masses. Malignancy in the absence of microcalcification was almost always invasive.

Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity.

Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting.

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.

Extended role for needle biopsy in the management of carcinoma of the breast.

Treatment choice in primary breast cancer is wide and still controversial; it seems likely that the optimum treatment for individual patients could be dictated by biological indicators of tumour behaviour. If biopsy could provide prognostic information as well as detailed tissue diagnosis then definitive treatment, with or without adjuvant systemic therapy, could be planned from the outset. We studied 140 patients with a clinical diagnosis of primary breast cancer to determine how much information could be obtained from Tru-Cut needle biopsies performed at the first clinic visit. Ten patients were found to have benign disease. Of 130 carcinomas, 123 (95 per cent sensitivity) were diagnosed correctly from the needle biopsies, with seven false negative and no false positive results (100 per cent specificity). Precise histopathology was predicted in 121 (93 per cent). Grade was correctly assessed in 77 of 112 (69 per cent), but needle biopsy was not accurate for assessment of lymphatic invasion nor elastosis. Steroid hormone receptors were assayed in 59 needle biopsies, and the incidence of oestrogen receptor positivity (34, 58 per cent) was similar to the resected tumours (35, 59 per cent), but the incidence of progesterone receptor positivity (26, 44 per cent) was lower (33, 56 per cent, P less than 0.04). Immunostaining with monoclonal antibody human milk fat globule membrane was accurate in the needle biopsies. DNA analysis by flow cytometry was performed in 37 tumours and the concordance between needle biopsies and resected samples was high. Tru-Cut needle biopsy obviates open biopsy and gives reliable detailed information.

The expression of milk fat globule antigens within human mammary tumours: relationship to steroid hormone receptors and response to endocrine treatment.

The value of steroid hormone receptors for the management of advanced carcinoma of the breast is often limited by the lack of availability of fresh tissue. Differentiation antigens may be estimated on paraffin-embedded fixed material by immunostaining, and the aim of this study was to determine whether staining with the monoclonal antibody raised to human milk fat globule (HMFG-1) could replace receptor measurements. The indirect immunoperoxidase technique was used to stain formalin-fixed paraffin-embedded tumour samples from 168 patients. All received tamoxifen or ovarian ablation as first-line systemic therapy, and all were evaluable for response (UICC criteria). One hundred and sixty-seven had oestrogen (ER) and progesterone receptors (PR) estimated. HMFG-1 staining was assessed as the percentage of tumour cells stained, and by the site of stain. The proportion of cells stained was highly correlated with both ER (P less than 0.0001) and PR (P less than 0.0001) and with response. When greater than or equal to 30% cells stained, 53 of 69 (77%) responded; when 20-29% stained 10 of 19 (53%) responded, when 10-19% stained seven of 19 (37%) responded, and when less than or equal to 9% cells stained 16 of 61 (26%) responded (P less than 0.0001). The median survival of patients with tumours that stained greater than or equal to 30% cells was 36 months, and with no cells stained, 11 months (P less than 0.0001). ROC (receiver operator characteristic) curves found that the optimum threshold for sensitivity and specificity of response prediction was greater than or equal to 20% cells stained. Cox's multiple regression analysis of 42 variables indicated that PR was the most important predictor of survival (P less than 0.000001), but that after PR the percentage of cells stained with HMFG-1 was the most important (P less than 0.0001). We conclude that immunostaining for HMFG-1 gives similar information to receptor status, and has the advantage that fixed archival tissue may be used.

The definition of the 'no change' category in patients treated with endocrine therapy and chemotherapy for advanced carcinoma of the breast.

In the criteria used for assessment of response to treatment for advanced breast cancer the definition of no change (NC) is clear; however, there is no indication of the duration of stabilization required for patients to qualify for this category of response. We have made the assumption that NC is a worthwhile category of response if the overall time to progression (TTP) and survival of this group is not significantly different from patients with partial remissions (PR). Two hundred and sixty-three evaluable patients treated with endocrine therapy and 302 evaluable chemotherapy-treated patients were studied and the TTP and survival curves for PR and periods of NC from 1 to 6 months compared. For the endocrine-treated patients the TTP and survival curves for NC became non-significantly different from the PR curves after 4 and 5 months respectively. For chemotherapy-treated patients the TTP curves became non-significantly different from PR at 4 months and for survival the period was 3 months. In order to define NC as a useful category of response and to eliminate the possibility that NC taken for a shorter period could simply represent a slowly progressive tumour, we suggest that the minimum period of disease stabilization be taken as 5 months for both endocrine- and chemotherapy-treated patients.

Expression of differentiation antigens within human mammary tumours is related to response to endocrine therapy and survival.

Human mammary tumours which are histologically well differentiated are more likely to synthesize receptors for estrogen (ER) and progesterone (PR) and to respond to systemic endocrine therapy. The aim of this study was to explore the relationship between differentiation, receptors and endocrine responsiveness in more detail by relating the expression of putative differentiation antigens within tumours to ER, PR and response to treatment. Sections of the primary tumours of 160 patients with advanced evaluable breast cancer were immunostained with 2 monoclonal antibodies (MAbs) (HMFG1 and HMFG2) raised against putative differentiation antigens found on the membranes which surround the milk fat globule. Tumours were highly heterogeneous with respect to antigen expression. However, the number of cells which expressed the antigens was highly correlated with ER and PR concentrations and with response to endocrine therapy. In tumours where greater than or equal to 20% of cells expressed the antigen recognized by HMFG1, 73% responded to endocrine therapy; this was similar to the response predicted by ER (67%) and PR (73%). Expression of HMFG1 was correlated with survival from the start of endocrine therapy (p less than 0.0001) to the same degree as ER and PR. Patients with tumours which expressed ER, PR and HMFG1 had the highest response rate (87%) and survival (median 49 months); the response in tumours which expressed none of these phenotypes was 13% and the median survival of these patients was 9 months. These results suggest that cells which express differentiation antigens also express ER and PR. Differentiated cells within mammary tumours may therefore be the target cells for systemic hormone, and also the source of factors which control tumour growth.

Ultrastructural observations on the basal lamina in the normal human breast.

The ultrastructure of the basal lamina of histologically normal human breast tissue was determined in 19 women undergoing operations for removal of a fibroadenoma or reduction mammoplasty. The day of the menstrual cycle was determined by hormone assay and direct questioning. Previously documented ultrastructural appearances were confirmed: in addition, three morphological variants were found. In all tissue examined, there was reduplication of basal lamina in some areas, which has been described previously as a pathological feature. Also, there was complex branching of the basal lamina into the periductular connective tissue. Some projections contained cytoplasmic processes and, in almost all, hemidesmosomes were seen. The third variant consisted of loops of basal lamina thrown up in folds into the collagenous stromal cuff. Reduplication of basal lamina was detected in breast tissue removed at all stages of the menstrual cycle, looping was not and could not be related to any particular phase of the menstrual cycle. However, complex branching was seen predominantly in the periovulatory and early luteal phase. We conclude that these appearances are normal variants of basal lamina. The appearance of branching basal lamina in the luteal phase suggests that this may be produced in response to endocrine stimulation.

Fibroblasts from relatives of patients with hereditary breast cancer show fetal-like behaviour in vitro.

Fetal and normal adult skin fibroblasts show distinctive migratory behaviour when plated on three-dimensional collagen gels. Skin fibroblasts from 13 of 15 patients with hereditary breast cancer showed fetal-like behaviour compared with only 1 of 12 age-matched healthy controls (p less than 0.015; Wilcoxon signed-rank matched-pairs test). In addition, 10 of 15 first-degree relatives of patients with hereditary breast cancer showed a fetal-like fibroblast phenotype, compared with none of 7 surgical controls (p less than 0.009; chi 2 test). These results suggest that abnormalities expressed by skin fibroblasts may help identify people at increased risk of breast cancer developing.

Effect of tamoxifen upon cell DNA analysis by flow cytometry in primary carcinoma of the breast.

The effect of tamoxifen upon cellular DNA ploidy in carcinoma of the breast was assessed by flow cytometry (FCM), in a prospective group of 77 patients with primary operable disease. Each had a needle biopsy at the outpatient visit for diagnosis and FCM analysis, and definitive surgery was performed a median of 8 days later. Forty received tamoxifen during this period - 40 mg qds loading dose for 24 h, followed by 20 mg daily until the day of operation: 37 patients received no therapy. The DNA histogram from the needle biopsy was compared with that obtained from the resected tumour for each individual. There was little change between the pair of histograms from tumours from the untreated patients. In those who had received tamoxifen the most consistent effect was a marked reduction in the magnitude of the 'tetraploid' peak in tetraploid or near-tetraploid tumours with DNA indices 1.8-2.0. There was little change in diploid or 'other DNA-aneuploid' tumours. In tetraploid tumours (DNA index of 2.0) the percentage of nuclei in the diploid S phase was significantly related to the percentage of nuclei in the diploid G2 + M/tetraploid G1 peak (P less than 0.003, unpaired t test). These data suggest that an effect of tamoxifen can be demonstrated by FCM upon tumours exhibiting a tetraploid or near-tetraploid DNA content. It is possible that tetraploid or near-tetraploid human mammary tumours may be a distinct group of endocrine responsive tumours within the overall group of aneuploid tumours, and that the majority are probably derived from the diploid population rather than being a true aneuploid population.

DNA analysis by flow cytometry, response to endocrine treatment and prognosis in advanced carcinoma of the breast.

The relationship between DNA content of mammary cancer and subsequent response to endocrine therapy was studied in 136 patients with advanced disease. All were treated with tamoxifen or ovarian ablation as first-line systemic therapy after relapse and were evaluable for response according to UICC criteria. DNA characterisation by flow cytometry was used on formalin fixed paraffin-embedded samples of tumour. Tumours were grouped according to DNA index into diploid (n = 52, 38%), 'tetraploid' (n = 46, 34%) and 'other DNA-aneuploid' (n = 38, 28%). The highest proportion of oestrogen receptor positive tumours (ER + ve) was found in the 'tetraploid' tumours (38/46, 85%, Chi-square = 8.53, P less than 0.02), and response rates, (SD + PR + CR), were 26/52 (50%), 34/46 (74%), and 15/38 (39%) respectively (Chi-square = 10.88, P less than 0.005). Patients with diploid or 'tetraploid' tumours survived longer and stayed in remission longer than those with 'other DNA-aneuploid' tumours. We suggest that 'tetraploid' or 'near tetraploid' human mammary tumours may comprise a distinct group of endocrine responsive tumours within the overall group of aneuploid tumours. The conventional interpretation of DNA histograms, grouping into diploid and aneuploid, may be masking important features of some tumour groups.

Endocrine therapy for advanced carcinoma of the breast: effect of tumor heterogeneity and site of biopsy on the predictive value of progesterone receptor estimations.

This study was carried out to assess the influence of site of biopsy and tumor heterogeneity upon the value of progesterone receptor (PR) measurements for prediction of response of patients with advanced carcinoma of the breast to tamoxifen or ovarian ablation. One hundred eighty-one assessable patients were studied. Sixty-nine % of responders and 31% of nonresponders were PR positive. The times to progression and survival were not significantly different for responders whether they were receptor positive or receptor negative. PR was measured on operable primary tumors (97), inoperable primary tumors (59), and secondary deposits (69). The proportion of responders who had PR-positive biopsies from these sites was 59%, 88%, and 61%, respectively, and the proportion of PR-positive nonresponders was 30%, 27%, and 42%, respectively. Ten to 12 separate PR measurements were made on seven tumors, and in four of them, there were PR-positive and PR-negative areas which could account for false positive and false negative results. We conclude that the optimum prediction of response was seen when the biopsy was performed on inoperable primary tumors. Errors in prediction of response at this site were, in part, explained by within-tumor heterogeneity of PR; the greater errors in prediction when measurements were made on operable primary tumors or on secondary deposits are presumed to be related to the additional effects of change in receptor status with time and between-tumor-site heterogeneity, respectively.

Endocrine therapy for advanced carcinoma of the breast: relationship between the effect of tamoxifen upon concentrations of progesterone receptor and subsequent response to treatment.

In some cell lines and tumors of mammary origin, tamoxifen causes an increase of progesterone receptor (PR) as a result of its partial estrogen agonist activity. In this study we have assessed the effect of tamoxifen on PR in patients with advanced carcinoma of the breast in order to test if those with a rise in PR are more likely to respond to endocrine therapy. PR was measured before and a median of 13 days after treatment with tamoxifen in a group of 52 patients with either locally advanced (n = 28) or recurrent (n = 24) carcinoma of the breast. Controls were a group of patients with operable disease who had two biopsies with no intervening tamoxifen (n = 51) or with intervening tamoxifen (n = 58). In the test group PR was higher in the second biopsy than the first in 21 patients, and 19 of these responded to continued endocrine therapy (90%). In the remaining 31 patients PR was either lower in the second biopsy (n = 19) or was negative in both biopsies (n = 12), and 11 of the total of 31 patients (35%) responded to continued endocrine therapy. The prediction of response and time to progression was better when both biopsies were taken into account than either the first or the second alone. The prediction of survival was similar for the group selected by an increase in the second biopsy and the group with PR present in the second biopsy. The controls without tamoxifen showed a marked variation in the level of PR in the first and second biopsies, suggesting heterogeneity of PR across the tumors studied. However, the PR level was significantly higher in the second biopsy in the controls given tamoxifen and in the test group compared with those with no intervening treatment (p = 0.031). This study indicates that some effect of tamoxifen upon PR can be demonstrated in human mammary tumors in vivo and that, by taking a second biopsy for PR estimation during treatment with tamoxifen, a more precise indication of subsequent response is obtained. The value of a single estimation of PR before treatment on secondary deposits is limited, and if one biopsy only is performed, it is of greater predictive value if taken after a few days treatment with tamoxifen.

Occurrence of a fetal fibroblast phenotype in familial breast cancer.

We have previously shown that fetal and adult human skin fibroblasts display distinctive migratory phenotypes when cultured on 3-dimensional collagen gels in vitro. In the present study, we have used this information to assess the migratory behavior of fibroblasts obtained from patients with either benign or malignant breast disease, and correlated this with the presence of a family history of breast cancer. We have observed that fibroblasts from 17/34 patients with no previous family history of breast cancer displayed fetal-type behavior in our assay system; in contrast, fibroblasts from 15/16 patients with a positive family history of breast cancer behaved abnormally. This apparently increased probability of expressing a fetal-type migratory phenotype in the patients with a family history is statistically significant (p less than 0.008). Skin fibroblasts obtained from 2 healthy and unaffected first-degree relatives (one male and one female) of patients with a family history of breast cancer also exhibited a fetal-type migratory phenotype.

Mammillary fistula.

During a 6-year period 38 patients with 52 mammillary fistulae were seen. On average, 5 abscesses had preceded recognition of a fistula in the areolar region (range 1-18). In 11 cases the fistulae were immediately apparent as discharging openings but they more commonly presented as subareolar abscesses. Fistulae were treated either by laying open or by excision of the track and at mean follow-up of 25 months (range 1-75) there has been recurrent fistula formation in only one case. Mammillary fistulae should be suspected and sought in all patients presenting with abscesses adjacent to the nipple.

Serum steroid binding proteins and the bioavailability of estradiol in relation to breast diseases.

Serum concentrations of sex hormone binding globulin (SHBG), corticosteroid binding globulin (CBG), and albumin were found to be normal in women with breast cancer (Ca), with benign breast disease (BBD), or with a family history of breast cancer (FHCa). Comparisons between serum steroid binding capacities and immunoassayable SHBG and CBG concentrations did not reveal abnormal forms of either protein. The serum distribution of estradiol (E2) was also determined, and women with Ca were found to have a significantly (P less than .025) higher mean percentage of non-protein-bound E2 than matched controls, but the difference was very small. In general, women with Ca also had proportionately more (P less than .05) albumin-bound E2 and less (P less than .05) SHBG-bound E2 in their sera than the controls, but the serum distributions of E2 in the BBD and FHCa subjects were the same as in controls. The dissociation rates of 5 alpha-dihydrotestosterone and E2 from SHBG in serum appear to increase with time in frozen serum samples, and this factor may effect measurements of the distribution of these steroids in serum.

Retroperitoneal tumour infiltration detected by bone scanning in patients with infiltrating lobular carcinoma of the breast.

Radionuclide bone scans performed in some patients with carcinoma of the breast showed abnormal retention of isotope in the renal pelvis which developed and progressed during the period of follow-up after mastectomy. This phenomenon was seen in 15 of 30 patients with infiltrating carcinoma of the breast (ILC) but in none of 29 with infiltrating duct carcinoma (IDC). Autopsies were performed in eight of the patients with ILC and three of those with IDC. Diffuse retroperitoneal and ureteric infiltration was seen in seven (88 per cent), with ILC and none with IDC. These data suggest that this scan abnormality is an indicator of retroperitoneal spread by ILC.