Histochemical studies with an estrogen receptor-related protein in human breast tumors.

The histochemical characteristics of a Mr 29,000 phosphoprotein related to estradiol receptor are described in a large series of human breast tumors. The antigen was detected with a monoclonal antibody (D5) raised against partially purified human myometrial estradiol receptor. An indirect immunoperoxidase method was used with methacarn-fixed, wax-embedded sections. Quantitation of staining and its reproducibility are described. Results with trucut biopsies agree with those obtained with larger tumor sections. Normal breast is infrequently positive. Histochemical staining is higher in invasive carcinoma than in normal breast with ductal carcinoma in situ adjacent to infiltrating tumors exhibiting intermediate values. Furthermore, most in situ carcinomas have a heterogeneous staining pattern. About 20% of invasive tumors also exhibit heterogeneity. No simple correlation is seen between staining and histological grade. There are more low-staining tumors in young (less than 50 yr old) patients than in older women. Staining correlates with levels of cytosol estradiol receptor but not cytosol progesterone receptor. However, cytosol estradiol receptor-negative, cytosol progesterone receptor-positive tumors tend to have positive Mr 29,000 phosphoprotein levels. Positive staining is associated with a higher response rate to hormone therapy (50%). None of the negative tumors responded to hormone treatment. With these patients, comparison of histochemical assay for Mr 29,000 phosphoprotein and [3H]estradiol binding assays indicated that the former was at least as good as the latter assay in predicting hormone response. About 20% of cytosol estradiol receptor-positive tumors have low Mr 29,000 phosphoprotein, and such tumors have poor response to hormone treatment.

Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cytometry.

Adjuvant systemic therapy for women with node-negative breast cancer is most easily justified for those patients at highest risk of relapse. We have examined the impact of tumor size, histologic grade, estrogen receptor (ER) status, tumor ploidy, and S-phase fraction (SPF) on relapse-free survival (RFS) for 169 patients with node-negative breast cancer in order to identify groups of patients at high and low risk of relapse. Patients with small tumors (less than or equal to 1.0 cm) had a significantly better RFS than those with larger tumors (P = .005), with 96% remaining relapse-free at 5 years. Patients with tumors less than or equal to 1.0 cm were thus excluded from analysis when attempting to define a group with a poor prognosis. Within the group of patients with tumors greater than 1.0 cm, tumor ploidy (P = .63), ER status (P = .3), or progesterone receptor (PgR) status (P = .24) did not predict for RFS. Patients with grade 1 or 2 infiltrating ductal tumors had a significantly better prognosis than those with grade 3 tumors (P = .04). The prognostic factor that gave the widest separation between subgroups, however, was SPF. Patients whose tumors were greater than 1.0 cm with an SPF less than or equal to 10% had a 5-year RFS of 78% compared with a 5-year RFS of 52% for those with an SPF greater than 10% (P = .006). We have combined tumor size and SPF to identify three prognostic groups: (1) tumor less than or equal to 1.0 cm, 5-year RFS 96%; (2) tumor greater than 1.0 cm plus SPF less than or equal to 10%, 5-year RFS 78%; 3) tumor greater than 1.0 cm plus SPF greater than 10%, 5-year RFS 52%. These prognostic groupings may help identify patients most suitable for adjuvant therapy.

The interaction of oestrogen receptor status and pathological features with adjuvant treatment in relation to survival in patients with operable breast cancer: a retrospective study of 2660 patients.

The oestrogen receptor (ER) status of 2660 patients with primary breast cancer has been related to the effect of different adjuvant systemic therapies on survival. However, as patients in the various treatment groups also had different prognostic features comparison between treatments was difficult. Over 90% of patients receiving tamoxifen (Tam) were postmenopausal compared with <20% of those receiving chemotherapy (CT). The latter had more positive nodes (85% vs 54%) and grade III tumours (54% vs 30%) than the Tam group. The combined CT and Tam group had similar characteristics to the CT alone group. The current reported increase in the proportion of women with ER+ tumours is explained by immunohistochemical analysis of ER and screening programmes. ER status was unrelated to survival in patients with small, low grade, node-negative tumours which was no different from that expected for age-matched women taken from the general population. The value of adjuvant treatment in these patients is therefore questionable. In those given any adjuvant treatment, survival of women with ER+ tumours was prolonged, with the greatest effect being seen in those receiving Tam. Patients with ER- tumours benefited from CT but the addition of Tam to CT improved survival only in those with ER+ tumours. ER status is now established as a major predictive factor for treatment selection in primary disease. Studies of prognostic and predictive markers may be invalidated by use of adjuvant therapy and selection criteria for different treatments. Survival will be influenced by both tumour biology and therapy. This important consideration must be remembered when analysing new markers, particularly in small studies.

Patterns of metastatic breast cancer in relation to histological type.

We have examined the clinical records fo 1238 patients with operable breast cancer to identify the sites of metastatic disease. Infiltrating ductal carcinoma (IDC) recurred more commonly in lung (P < 0.05), pleura (P < 0.05) and brain (P < 0.05), while infiltrating lobular carcinoma (ILC) more commonly metastasised to the bone marrow (P < 0.01) and peritoneum (P < 0.01). Bone involvement as the initial presentation of distant metastatic disease occurred in over 50% of women with ILC, significantly more commonly than in those with IDC (34%, P < 0.01). Survival was similar for the two groups, both from time of diagnosis and from time of development of distant metastases.

Cyclical tumour variations in premenopausal women with early breast cancer.

The hormonal milieu at the time of tumour excision may have a significant impact on survival in premenopausal patients with breast cancer, with those undergoing surgery between days 3 and 12 of the menstrual cycle having a worse prognosis. To investigate possible mechanisms which might explain this finding, histological features of tumours from 363 patients included in two studies from Guy's Hospital have been reviewed. Axillary nodal involvement occurred in 71/115 (62%) of patients whose primary tumour was excised between days 3 and 12 of the cycle, compared with 116/248 (47%) of patients undergoing surgery at other phases of the cycle (chi 2 = 7.04, P < 0.01). Vascular invasion was observed in 54/115 (47%) of primary tumours removed between days 3 and 12 and 82/248 (33%) of tumours removed at other times (chi 2 = 6.47, P < 0.02). Multivariate analysis of factors influencing survival indicated that both axillary nodal status and phase of the cycle were highly significant independent predictors of prognosis.

c-erbB-3 protein expression in ductal carcinoma in situ of the breast.

c-erbB-3, A recently identified member of the type I tyrosine kinase receptor family, has been shown to be overexpressed in invasive ductal carcinoma of breast. In this study, expression of the c-erbB-3 protein was examined in 57 cases of pure ductal carcinoma in situ of the breast (DCIS) by immuno-cytochemical methods. Staining was either absent (17 cases), present at levels equivalent to that found in adjacent normal tissue (20) or greater than in normal tissue (20). In most cases the pattern of staining was cytoplasmic, but in 4 cases with the most intense reaction there was also focal membrane staining. In the same series of cases, c-erbB-2 protein had previously been shown to be overexpressed in 28 of 57 cases, c-erbB-2 overexpression was correlated with normal level of c-erbB-3, and lack of c-erbB-2 expression was correlated with c-erbB-3 overexpression.

Increased use of immunohistochemistry for oestrogen receptor measurement in mammary carcinoma: the need for quality assurance.

This paper outlines the changes which have occurred over the last 25 years in the methods employed for the measurement of oestrogen receptors to aid the management of women with breast cancer. Immunohistochemistry is now the method of choice and knowledge of oestrogen receptor status is being used with increasing frequency for the selection of adjuvant treatment as well as for the treatment of metastatic disease. It is essential that good quality assurance procedures are established so that results are reproducible and can be used with confidence in individual centres as well as being comparable with those produced elsewhere. A retrospective study of 170 women with metastatic breast cancer provides the basis for a discussion on the advantages and pitfalls of the immunohistochemical assay. Particular emphasis is paid to the choice of cut-off and how the results may be applied in patient management.

Tumour grade does not change between primary and recurrent mammary carcinoma.

The primary tumour grade in 115 patients with infiltrating ductal carcinoma of the breast was compared with the type of the ductal carcinoma in situ (DCIS) component and with the grade of 169 locally recurrent and metastatic lesions. 102 patients had axillary lymph node metastases at the time of primary surgery, 49 had subsequent recurrences and 36 had both. There was concordance of grade between the primary tumour and axillary lymph node metastases and with subsequent locally recurrent and metastatic lesions. The type of the DCIS component was also significantly associated with the grade of the infiltrating component. No evidence of progression of tumour grade between these phases of mammary carcinoma was found.

Proliferative activity, histological grade and benefit from adjuvant chemotherapy in node positive breast cancer.

The influence of S-phase fraction (SPF), measured by DNA flow cytometry, and histological grade on outcome following adjuvant chemotherapy was analysed for 214 patients with node positive breast cancer treated at Guy's Hospital who were entered into the Guy's/Manchester trial of combination chemotherapy with cyclophosphamide/methotrexate/5-fluorouracil (CMF) vs. no adjuvant treatment. Adjuvant CMF significantly improved relapse-free survival (RFS) for premenopausal patients whose tumours had an SPF of 10% or less (control vs. CMF, P = 0.05) and premenopausal patients whose tumours had an SPF over 10% (control vs. CMF, P = 0.003). No significant improvement in RFS attributable to CMF was seen for either subgroup of postmenopausal patients. When patients were divided into subgroups based on histological grade of tumour, an improvement in RFS attributable to CMF was seen for premenopausal patients with well differentiated (grade 1 or 2) tumours (control vs. CMF, P = 0.03) and premenopausal patients with poorly differentiated (grade 3) tumours (control vs. CMF, P = 0.006). Again, no improvement in RFS was noted for any subgroup of postmenopausal patients defined by tumour grade.

Overexpression of the c-erbB-2 oncoprotein: why does this occur more frequently in ductal carcinoma in situ than in invasive mammary carcinoma and is this of prognostic significance?

Overexpression of c-erbB-2 occurs in 60% of in situ and 25% of infiltrating ductal carcinomas. We have previously found very strong associations between immunohistochemical staining for c-erbB-2 and histological pattern and nuclear size in ductal carcinoma in situ (DCIS) and less strong correlation with proliferative activity. In a further study of infiltrating ductal carcinomas we have found that, in addition to tumours arising from c-erbB-2 positive, large celled, rapidly proliferating, comedo carcinomas and c-erbB-2 negative small celled cribriform/micropapillary carcinomas with a low proliferative rate, there is a third group of c-erbB-2 negative tumours with large nuclei and variable proliferative activity. These latter tumours are not seen in pure DCIS suggesting that they have a very transient in situ stage. Therefore, although in pure DCIS c-erbB-2 positively appears to be associated with tumours with a greater invasive potential, and c-erbB-2 negativity with tumours having a more favourable prognosis, the latter is not necessarily true in infiltrating disease.

Immunochemical analysis of the p53 oncoprotein in matched primary and metastatic human tumours.

There is much interest in the range of genetic aberrations which occur in human malignancies. An immunohistochemical study has been carried out to investigate the consistency of expression of abnormally accumulated p53 protein in paired samples of archival primary and metastatic carcinomas. The staining of methacarn-fixed tissue from 136 matched pairs of mammary carcinoma and 20 cancers from other sites was completed using antibody CM-1 and DO1 in a sensitive peroxidase-conjugated streptavidin-biotin technique. The majority of tumour cells were positive in 25% and the tumours were negative in 17% of the primary carcinomas; staining was heterogeneous in the remaining cases. Staining was identical in 180/186 (96%) metastatic lesions. An ELISA assay carried out on 12 matched pairs of the tumour specimens demonstrated that altered conformation of the aberrant p53 protein present in a primary lesion was maintained in its metastasis. These data indicate that alterations in the p53 gene result in a relatively stable phenotype and that progression of disease is not usually accompanied by either further mutation or loss of the mutant allele.

Acantholytic variant of squamous-cell carcinoma of the breast.

Three cases of acantholytic squamous-cell carcinoma of the breast are reported. They all had histological features resembling those of angiosarcoma or adenocarcinoma. They were not angiosarcoma, since in all three cases areas of squamous differentiation were present; in addition, the neoplastic cells were negative when stained for factor VIII, but were positive with anti-epidermal keratin. The glandular pattern exhibited, especially in Case 2, was difficult to differentiate from that of an ordinary carcinoma. However, the presence of dyskeratotic cells within the lumina, and the absence of alcian blue/periodic acid-Schiff positive material, and epithelial membrane antigen staining, were evidence against the diagnosis of adenocarcinoma. The patients died 5, 6, and 16 months after the diagnosis. Tumors with these histological features may have a very aggressive clinical course.

Microglandular adenosis, apocrine adenosis, and tubular carcinoma of the breast. An immunohistochemical comparison.

Four cases of microglandular adenosis (MA), together with four cases of apocrine adenosis (AA) and 10 cases of tubular carcinoma (TC) of the breast were studied at the light and immunohistochemical level. One case of MA was studied with electron microscopy. MA is characterized by an absence of myoepithelial cells (ME), epithelial membrane antigen (EMA), and gross cystic disease fluid protein (GCDFP-15). The absence of EMA in MA makes it unique among benign glandular hyperplasias of the breast. AA contains myoepithelial cells and a distinct basal lamina. It is characterized by the presence of GCDFP-15, the specific apocrine marker, which is not present in MA. TC lacks both myoepithelial cells and a basal lamina. It is negative for GCDFP-15. Periductal and vascular elastosis are common and usually prominent, whereas they are not found in either MA and AA. Other stromal changes further distinguish the three lesions. These three distinct entities can be separated objectively and unequivocally and it is essential that this be done so as to prevent confusion.

Ductal adenoma of the breast--a review of fifteen cases.

The term ductal adenoma has been recently introduced to describe a solid benign lesion of breast ducts. This study describes the clinical, morphologic, and immunohistochemical features of 15 cases of ductal adenoma. Ductal adenomas are usually single, occasionally multiple, lesions occupying medium- and large-sized breast ducts. They may occur in women of all ages, although the majority of patients are 60 years of age or greater. Ductal adenomas usually present clinically as breast lumps which may mimic carcinoma; less commonly, they are associated with nipple discharge. Patients in this series showed no family or previous history of breast disease and had uneventful follow-up after local excision. Despite often showing worrying pseudoinfiltration and cytologic atypia, the immunohistochemical demonstration of a myoepithelial layer and intact basement membrane around the tubules was clear evidence of the benign nature of the lesions. We conclude that most ductal adenomas evolve by sclerosis of benign intraduct papillary lesions, although processes similar to sclerosing adenosis and, possibly, duct ectasia may contribute to the pathogenesis of a proportion of cases. It is hoped that a wider appreciation of the entity of ductal adenoma will reduce the diagnostic uncertainty that continues to surround these and related lesions.

Immunohistochemical demonstration of c-erbB-2 protein in mammary ductal carcinoma in situ.

The presence of the c-erbB-2 protein, demonstrated immunohistochemically with antibody 21N, has been studied in 72 cases of pure mammary ductal carcinoma in situ. Sixty-one percent of cases showed positive staining. The protein was always present in large-cell comedo type of ductal carcinoma in situ, and never in small-cell cribriform/micropapillary type of ductal carcinoma in situ. When nuclear size was measured, staining was associated with tumors containing cells with large nuclei measuring up to 20 mu, and was never present in lesions containing cells with small nuclei measuring 10 mu or less. In tumors of mixed histopathologic type of ductal carcinoma in situ, variable staining was seen; the cells with small nuclei never stained, while the majority of cells with large nuclei reacted positively. The possible relevance of these findings to the biologic behavior of DCIS is discussed.

Immunohistochemical detection of p53 protein in mammary carcinoma: an important new independent indicator of prognosis?

In an immunohistochemical pilot study of 195 primary breast cancer patients with a 10-year median follow-up we found that patients with carcinomas who express p53 protein in the majority of their tumor cells (19% of the cases) have a considerably worse prognosis than those who do not. The effect of the presence of the protein is seen on disease-free interval (chi-square, 11.69; P < .001), overall survival (chi-square, 19.68; P < .001), and survival after relapse (chi-square, 4.93; P < .02), and is seen in node-negative (chi-square, 6.99; P < .009) and node-positive (chi-square, 13.05; P < .001) patients. Furthermore, the effect is most apparent in patients with infiltrating lobular and grade II infiltrating ductal carcinomas (chi-square, 27.97; P < .001) that have a rather heterogeneous clinical behaviour and are difficult to subdivide on the basis of currently available markers. Cox multivariate analysis shows that p53 majority staining is second only to node status in significance of effect on overall survival.

Breast lymphomas: a clinicopathologic review.

Primary lymphoma is an uncommon tumor in the breast. Review of the literature shows two distinct clinicopathologic groups. One, which affects young women, is frequently bilateral, is often associated with pregnancy, and is a Burkitt-type lymphoma. The second group affects older women, is usually B-cell non-Hodgkins-type lymphoma, and presents clinical features identical to carcinoma of the breast. Three recent studies have suggested that up to half of the cases in the latter group belong to the category of lymphomas arising from the mucosa-associated lymphoid tissues. We have identified nine cases of primary lymphoma from the files of Guys Hospital Clinical Oncology Breast Unit in the 16-year period from 1974 to 1990. The clinical features have been reviewed and the tumors have been evaluated both on a morphologic and an immunohistochemical basis, and seven of nine of the cases have been screened for t[14;18] translocation using the polymerase chain reaction. All the tumors occurred in women older than 50 years and who presented with features of mammary carcinoma. One tumor was true histiocytic lymphoma; the remaining eight cases were B-cell lymphomas. Seven of the latter cases were high-grade B-cell lymphomas and one was a true follicular lymphoma. None of our cases showed the features of lymphoma arising in mucosa-associated lymphoid tissue.

Prediction of local recurrence of ductal carcinoma in situ of the breast using five histological classifications: a comparative study with long follow-up.

The increased detection of ductal carcinoma in situ (DCIS) by mammographic screening and the more widespread use of breast-conserving surgery have led to a search for histological features associated with the risk of recurrence. In a case control study of 141 patients with long follow-up, we compared the ability of five morphological classifications to predict recurrence after local excision. A significant correlation was not found between recurrence and growth pattern when a traditional classification based on architecture was used nor with necrosis when a scheme based principally on this feature was employed. A correlation was, however, found between recurrence and "differentiation" as defined by nuclear features and cell polarization in a classification recently formulated by the European Pathologists Working Group (EPWG), but this failed to reach statistical significance at the 5% level. A stronger and statistically significant correlation was found between nuclear grade as defined by the EPWG and recurrence when cell polarization was disregarded, using the classification currently employed by the UK National Health Service and European Commission-funded Breast Screening Programmes. This was attributable to a small number of recurring cases being downgraded as a consequence of exhibiting polarized cells. A significant correlation between histology and recurrence was also observed using the Van Nuys classification, which is based on nuclear grade and necrosis. Whether the tumor recurred as in situ or invasive carcinoma was unrelated to histological classification, as was the time course over which it occurred. These findings strongly support the use of nuclear grade to identify cases of DCIS at high risk of recurrence after local excision, but further work is necessary to determine whether nuclear grade or necrosis is more appropriate to subdivide the non-high-grade cases.

Relationship of blood prolactin levels and the risk of subsequent breast cancer.

Between 1968 and 1976 a total of 5162 women volunteers were enrolled into a prospective study conducted on the Island of Guernsey. Up to February 1990 145 women subsequently developed breast cancer. Blood samples were taken at the time of enrollment and prolactin levels were known for 85% of the volunteers. In calculating the relationship between blood prolactin levels and subsequent breast cancer risk, women were excluded if they had a hysterectomy or an oophorectomy or had cancer at any site before enrollment. The final analysis was based on 2596 premenopausal and 1180 naturally postmenopausal women and, of these respectively, there were 71 and 40 volunteers who subsequently developed breast cancer. The total follow-up for these two groups was 49,941 and 22,360 woman-years, respectively. In assessing the relationship between blood prolactin levels and risk of subsequent breast cancer the cohort was divided into quintiles according to prolactin concentration and relative risks (RR) were estimated. In calculating these values possible confounding by age at entry, age at menarche, parity, age at first birth, years since menopause, body build, history of benign breast disease and family history of breast cancer were taken into consideration. There was no significant relation between risk of breast cancer and prolactin in either pre- or postmenopausal women. Hence prolactin appears not to be an important determinant of breast cancer risk.

Adenomyoepithelioma of the breast with malignant features.

The clinico-pathological features of 7 cases of adenomyoepithelioma of the breast with features suggestive of malignancy are presented. There was a high incidence of local tumour recurrence, in 2 cases as high-grade infiltrating carcinoma of the breast of no special type ("ductal", grade III). One patient died as the result of a clinically diagnosed cerebral metastasis. Histological examination of the primary breast tumours reveals two main patterns: (1) tumours consisting in part of typical adenomyoepitheliomas but which merge with areas of obviously invasive malignant cells and (2) neoplasms that have the overall architecture of an adenomyoepithelioma but which, on close examination, are found to contain foci of cellular atypia and increased mitotic activity. The two patterns of tumour exhibit the same clinical behaviour and should be distinguished from adenomyoepitheliomas, which are cytologically bland throughout.