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The polymerase chain reaction (PCR) is a powerful tool to examine genetic alterations in tumor samples. We describe a simple, rapid, nonisotopic PCR method to semi-quantitatively determine the number of gene copies in human formalin-fixed paraffin-embedded tissue. The procedure is exemplified by analysis of 15 years old breast cancer samples to determine the presence of amplification of c-erb-B2. The samples were obtained from routine specimens kept in pathological archives. Patients with amplified samples showed a poor prognosis, both for recurrences and death in breast cancer.

Immunohistochemical analysis with the monoclonal antibody (MoAb) A-80, recognizing a tumor-associated cytoplasmic mucin-type glycoprotein, and cytometric nuclear DNA assessment were performed on 314 surgical specimens of the human mammary gland. The series included 36 benign conditions, 34 epithelial hyperplasias, 40 carcinomas in situ, and 204 primary invasive carcinomas. Normal breast parenchyma, benign tumors, and other nonmalignant lesions were all of DNA diploid (euploid) type and rarely expressed the A-80 glycoprotein. Differences in MoAb A-80 immunoreactivity and nuclear DNA content were noted among subtypes of epithelial hyperplasias. Fifteen of 34 epithelial hyperplasias were of DNA aneuploid type and the majority were A-80 immunoreactive. Of these 15 immunoreactive aneuploid epithelial hyperplasias, atypical intraductal hyperplasia was the most common subgroup. None of the 19 epithelial hyperplasias of DNA euploid type immunoreacted. Most of the intraductal (33 of 40) and invasive (180 of 204) carcinomas immunostained with MoAb A-80. The majority of the A-80 immunoreactive malignant tumors were of DNA aneuploid type (26 of 33 carcinomas in situ and 108 of 180 invasive mammary carcinomas). The results suggest that expression of the A-80 glycoprotein occurs at an early stage of malignant transformation. Genetically stable (euploid) mammary tumors seem to immunoreact with MoAb A-80 less frequently than genetically unstable (aneuploid) tumor variants. Combined analysis with MoAb A-80 and of nuclear DNA content in premalignant and malignant mammary lesions could be a useful tool of differential diagnostic and prognostic value.

The authors studied prognostic factors in 77 patients with primary cystosarcoma phyllodes (CSP) of the breast. Median patient age was 50 years of age, and the median follow-up time was 8 years. Sixteen patients (21%) had distant metastases and subsequently died of CSP. Clinical variables such as age, symptom duration, clinical tumor size, and type of surgery were not of prognostic value. Local recurrence was more common among patients treated with breast-conserving surgery than among those treated with mastectomy. However, there was no significant difference between these two subgroups in terms of distant metastasis-free survival or overall survival. The prognostic significance of several histopathologic parameters was also assessed, e.g., stromal cellularity, stromal cellular atypism, mitotic activity, atypic mitoses, stromal overgrowth, tumor contour, tumor necrosis, and heterologous stromal elements. In a multivariate Cox analysis, the only features that were found to be independent prognostic factors were tumor necrosis (P less than 0.05) and presence of stromal elements other than fibromyxoid tissue (P less than 0.01). In summary, additional studies of prognostic factors in CSP are warranted because of the conflicting results in published reports.

Between November 1, 1976 and December 31, 1981, 826 post-menopausal females with operable breast cancer were included into a trial comparing tamoxifen 40 mg daily for 2 years with no endocrine treatment. Patients without axillary lymph node metastases and tumors less than 30 mm received no other treatment whilst those with more advanced disease were in addition superrandomised to receive postoperative irradiation or 12 courses of CMF. With a mean follow-up of 44 months tamoxifen significantly reduced the incidence of recurrence. There was no significant interaction between the effect of tamoxifen and any other treatment or prognostic subgroup.

The frequency of new primary cancers was studied in 1846 postmenopausal patients included in a randomised trial of tamoxifen as an adjunct to primary surgery for early breast cancer. The median follow-up was 4.5 years (range 0.5-10.5 years). The number of new cancers in the tamoxifen group (n = 57) did not differ significantly from that in the control group (n = 70). However, in tamoxifen patients second breast cancers occurred less often and endometrial cancer occurred more often than in the controls. The increase in endometrial cancers was probably related to the agonistic oestrogenic effects of tamoxifen and was most pronounced in those treated for over 2 years.

The prognostic influence in operable mammary carcinoma of morphologic predictors evaluated in smears of needle-biopsy aspirate and in tissue sections and of clinical stage was investigated. Size of nuclei in smears of aspirate was found to convey prognostic information of the same order of importance as the variables tubule information and mitotic frequency in tissue sections. The combination of clinical stage with variables in smears and in sections enhanced the explanatory value.

The grade of nuclear atypia (low, medium, or high) was compared with DNA content in 122 tumors comprising 73 serous ovarian tumors and 49 mammary carcinomas of ductal type. The study was performed on two consecutive histologic sections 4 mu in thickness, one of which was stained with hematoxylin-eosin for estimation of nuclear atypia, and the other with Feulgen for measurements of DNA. In both tumor groups there was a significant correlation between nuclear atypia and nuclear DNA content. The results suggest that DNA analysis is of significant additional prognostic value even when the grade of nuclear atypia is known.

In a randomized trial, 960 women with Stage 1-3 operable breast cancer were treated by a modified radical mastectomy alone, or by the same procedure, preceded or followed by radiotherapy (4500 rad to the breast/chest wall, and internal mammary, axillary and supraclavicular lymph nodes). Up to ten years after treatment, there is an increasing gap between the recurrence-free survival of the irradiated patients and the surgical controls. Between the two types of radiotherapy, there was no difference. There were significantly fewer distant metastases and a tendency for improved survival in node positive patients treated with postoperative radiotherapy, compared to the surgical controls, this difference was, however, statistically not significant.

The relationship between DNA distribution pattern and estrogen receptor (ER) content was investigated in primary mammary carcinomas from 56 female patients. The data showed that mammary carcinomas in which the DNA amounts of the tumor cells do not deviate conspicuously from diploid or tetraploid DNA values of normal mammary epithelium are characterized by high levels of ER. On the other hand, mammary carcinomas containing a sizable number of cells with DNA amounts in the normal DNA synthesis region or with high degrees of aneuploidy are distinguished by low or even unmeasurable ER levels. These results strongly indicate a correlation between nuclear DNA distribution patterns and ER levels in primary breast carcinomas.

A series of 203 consecutive patients with operable carcinoma of the breast was analysed with regard to correlations between a set of immunologic and clinical variables existing at the time of the diagnosis. No major correlations were revealed between immunologic variables on the one hand and clinical features or the course of the disease on the other. The well-known prognostic relevance of tumour size, involvement of the axilla and the histological grade of malignancy was evident.

During 1958, 91 cases of breast carcinoma in women below 30 years of age were reported to the Swedish Cancer Registry. A histologic reexamination showed that 16 of these women had only benign proliferative conditions. Among the remaining 75 patients, two had noninvasive carcinoma, eight advanced carcinoma, and 65 operable invasive carcinoma. The carcinomas were characterized by little fibrous stroma and little or no elastosis. The survival rate of the patients with invasive breast carcinomas was similar to that in a total geographic material of all ages with breast carcinoma. The carcinoma was treated during pregnancy or within 1 year after delivery in 15 patients, with the same survival rate as for those whose carcinoma was not complicated by pregnancy.

The nuclear DNA content in tumor cells from invasive ductal breast carcinomas was analyzed in 26 patients who survived more than 10 years and in 23 patients who died within 2 years after operation. The DNA content of individual tumor cells was measured in sections from the originally paraffin-embedded specimens. In short-term survivors, a large proportion of cells with very high DNA values was found in all tumors except one. Only two patients of the long-term survivors had tumors that exhibited such high DNA values. Prognostic information obtained by DNA analysis compared with histologic malignancy grading showed that the specificity using DNA analysis was distinctly higher. The data thus suggest that analysis of DNA content of tumor cells in breast adenocarcinomas can be a useful supplement to histologic malignancy grading to obtain prognostic guidance in individual patients.

Immediately after mastectomy, estrogen receptor concentration was measured in fine needle aspirates and in histologic tissue specimens from 13 human breast carcinomas. Fine needle aspirates were shown to contain enough material for receptor analysis when the sensitive method of isoelectric focusing in slabs of polyacrylamide gel was used for receptor determination. Aspirations with two different needles (outer diameter 0.7 and 1.4 mm) from each tumor yielded highly correlated receptor values (r = 0.97, p less than 0.0001). There was also a highly significant correlation (r = 0.91, p less than 0.0001) between the receptor content in needle aspirates and in tissue specimens from the same tumor.

The simultaneous prognostic influence of multiple clinical and microscopic features of mammary carcinoma was analysed in 581 women with radical mastectomy. The most important of these features were connected with the extent of the disease in the axillary lymph nodes. In substantial groups of patients, however, a favourable outcome could be predicted from microscopic features of the primary tumour, viz. tubule formation, mitotic frequency and elastosis.

Histological specimens from 80 invasive breast carcinomas comprising typical cases of 16 ductal, nine papillary, 14 comedo, 13 colloid (mucous), 15 lobular, and 13 medullary carcinomas were examined with respect to nuclear DNA and estrogen receptor content. In agreement with previous studies, ductal carcinomas were found to exhibit different types of nuclear DNA distribution patterns, i.e., tumors with DNA values in the normal diploid or tetraploid regions indicative of good prognosis (euploid tumors) or those with values exceeding the normal tetraploid region indicative of poor prognosis (aneuploid tumors). The majority of the papillary and colloid tumors were euploid, while comedocarcinomas in general had aneuploid profiles. These findings are in agreement with expected survival within these patient groups. In lobular breast carcinomas, the correlation between the DNA distribution patterns and expected patient survival was less obvious; and in medullary carcinomas where the vast majority of the tumors showed aneuploid DNA profiles, the correlation to expected patient survival was low. Thus, lobular carcinoma in general seems to have a worse prognosis than is expected from nuclear DNA analysis, whereas medullary carcinomas in general seem to carry a better prognosis than indicated from DNA measurements. In agreement with earlier reports there was a good correlation between nuclear DNA content of the tumor cells and cytosol estrogen receptor values, i.e., euploid tumors in general exhibited relatively high receptor levels, whereas aneuploid tumors had low or unmeasurable estrogen receptor levels.

This report provides a histological review of 264 female primary breast cancers analyzed for estrogen receptor protein (ER). We also describe 5 cases of male breast cancer all of which bound estradiol specifically. Generally the ER concentrations were lower in the tumors of premenopausal women than in those of postmenopausal women. Three types of cancer with specific morphological features were shown to have ER concentrations that differed significantly from the other types of cancer. Medullary and comedo carcinoma had very low and papillary carcinoma very high levels of ER. No obvious trend in ER concentration was found in unspecified ductal carcinoma, colloid carcinoma, nor in lobular carcinoma. A positive correlation was demonstrated between ER content and degree of differentiation in ductal carcinoma. Cancer with lymphoid infiltration generally showed low ER levels.

Isoelectric focusing in polyacrylamide gel combined with limited proteolysis is a simple and specific method for quantitation of estradiol receptors in breast cancer tissue. At least eight different samples can be analyzed simultaneously on one gel, and the whole procedure, including sample preparation, takes less than 7 hr. In comparison with sucrose gradient centrifugation, isoelectric focusing is more sensitive, possibly due to the short time (1.5 to 2 hr) needed for the analysis. Furthermore, only one incubation with tritium-labeled estradiol is needed for an analysis, which means that a smaller amount of tumor tissue is needed than for most other methods. This fact allows analysis of the estrogen receptor content in tumor material obtained from fine-needle biopsy.

A randomized trial of preoperative radiotherapy in operable breast cancer was conducted from 1971 to 1976. The diagnosis was established by fine-needle aspiration biopsy. A dose of 4500 rad over five weeks was given to the chest wall, the breast and the lymph nodes of the axilla, the supraclavicular fossa and the internal mammary chain. Modified radical mastectomy was performed six weeks or more after completed radiotherapy. In control patients the same operation was performed without prior radiotherapy. By random allocation, one control group received no further treatment and postoperative irradiation was given to the other controls. Preoperative radiotherapy reduced the incidence of local and regional recurrence and of distant metastases, and also the mortality, as compared with the surgery only group. Postoperative radiotherapy as given in this trial gave almost equal reduction of local and regional recurrence but did not diminish the frequency of distant metastases or the mortality.

Stereotaxic needle biopsy was performed on 121 radiographically demonstrated non-palpable breast lesions, which were excised within 3 months after the needling. Of the 62 histopathologically proven malignant tumors, malignant or probably malignant epithelial cells were obtained at needle biopsy, in about 80 per cent of cases, in which the cellular material was quantitatively adequate. There was no false positive cytologic result. The combined radiologic and cytologic assessment gave a correct diagnosis in 61 of the 62 histopathologically malignant tumors. There was metastatic tumor involvement of the lymph nodes in only 3 of the malignant tumors.

The extent of radiation induced immune suppression was analysed in 100 patients with carcinoma of the breast. The relative changes of lymphocyte counts and stimulations by PHA and PPD were similar in patients who differed with regard to age, size of tumour and its malignancy grade or axillary node condition. Moreover, no difference in the degree of radiation induced immune suppression existed between patients who developed recurrent disease and those who remained free of disease during a follow-up period of 4 1/2 to 7 years.

At the time of treatment in 1977 for primary breast cancer of all stages, the tumors of 270 women were examined for estrogen-receptor (ER) content by isoelectric focusing on polyacrylamide gel. The procedures for the initial and follow-up examinations were standardized. Postoperative adjuvant radiotherapy or chemotherapy was given to pre- and postmenopausal women aged up to 70 years with nodal involvement. Adjuvant tamoxifen treatment (20 mg twice daily for 2 years) was given to one-half of the postmenopausal patients aged 70 years or less, selected at random, and irrespective of any other adjuvant therapy. These forms of treatment appeared not to introduce bias into the results of the study. There was no correlation between the ER content and tumor spread. Symptom-free survival during the follow-up period was best for the patients with tumors with a high ER content, poorer for the patients with tumors of intermediate ER content and least favorable for patients with tumors having low ER content. (Ranges for high, intermediate and low levels: 2.18-9.08, 0.13-2.17 and 0-0.12 fmoles/microgram DNA). The differences between the survival curves were significant. For the 120 operable patients with nodal involvement there were also significant differences in recurrence-free survival. The premenopausal patients in this group likewise showed a significant difference in survival with respect to ER content. For the operable patients with nodal involvement and poorly differentiated tumors there was also a difference in the survival rate depending on the ER content. In this study the ER content of breast cancer was shown to be a quantitative rather than a qualitative entity. The study confirms the independent prognostic importance of the ER content and points to its potential value as a stratification criterion in adjuvant therapy trials.