Demonstration of progesterone receptors in paraffin wax sections of breast carcinoma.

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.

Estrogen receptors in ductal carcinoma in situ of breast.

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.

Predictors of local recurrence following conservative breast surgery and radiation therapy. The influence of tumor size.

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.

Imprint immunocytochemistry of estrogen receptors in breast carcinoma.

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.

Salivary duct carcinoma (cribriform salivary carcinoma of excretory ducts). A clinicopathologic and immunohistochemical study of 12 cases.

Salivary duct carcinoma (cribriform salivary carcinoma of the excretory ducts [CSCED]) is an uncommon malignant tumor which occurs predominantly in men (83% in this series; mean age, 61 years) and most often in the parotid gland (92% in this series). The outcome is unfavorable for most patients; of 11 of 12 patients with follow-up, 45% had local recurrence, 54% had distant metastasis, and 45% were dead of disease within 10 years of diagnosis (mean, 3 years). Metastases to lymph nodes were common (72%). Immunohistochemical studies on paraffin-embedded tissue revealed that most tumors reacted with antibodies known to mark adenocarcinoma: B72.3 (11 of 11) and Lewis Y (ten of ten). High and low molecular weight cytokeratins were present in most tumors (nine of ten and seven of nine cases, respectively), supporting the concept that these adenocarcinomas were of ductal origin. Parotid ducts adjacent to CSCED expressed B72.3 in six of nine cases studied, but parotid ducts from normal tissue (adjacent to benign mixed tumors or enlarged periparotid lymph nodes) rarely expressed this marker (one of 17 cases). The detection of B72.3 diffusely in parotid ducts, especially those with atypia, may imply the presence of malignant tumor nearby, which could be useful in evaluating limited tissue from the parotid. However, further studies are necessary to confirm the significance of this finding.

Flow cytometric and histological analysis of ductal carcinoma in situ of the breast.

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.

Florid papillomatosis of the nipple: immunohistochemical and flow cytometric analysis of two cases.

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)

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses.

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.

Pancreatic microcystic adenoma coexistent with pancreatic ductal carcinoma. A report of two cases.

We report two cases of microcystic (glycogen-rich) adenoma of the pancreas with coexistent pancreatic adenocarcinoma. Both patients presented with an epigastric mass. On laparotomy, each had two separate pancreatic tumors. The benign tumors were composed of small cysts with a flattened to cuboidal glycogen-rich epithelium. Both malignant tumors were composed of mucinous epithelium and showed positive staining for CEA and Leu-M1. Although pancreatic microcystic adenoma and ductal adenocarcinoma are believed to arise from different precursor cells, the association reported here suggests a common predisposition to both tumors. Careful examination of the pancreas is warranted in cases of microcystic adenoma to rule out a possible coexistent pancreatic carcinoma.

A prospective comparison of DNA quantitation by image and flow cytometry.

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.

Nucleolar organizer regions and Ki-67 immunostaining in ductal breast cancer: a comparative study.

53 cases of invasive ductal (NOS) carcinomas of the breast were studied by means of an immunostaining method with Ki-67 monoclonal antibody and an argyrophilic method for the demonstration of Nucleolar Organizer Regions (AgNORs). The percentage of cancer cells with nuclear Ki-67 immunoreactivity and the mean number of NORs for each tumour were statistically related. The data obtained showed a good correlation between Ki-67 index and NOR score (rS = 0.47 - P less than 0.001). The authors suggest that the AgNOR method--which is applicable to routinely processed material--might effectively substitute Ki-67 immunostaining as a marker of cell proliferation in ductal breast cancer.

Epidermal growth factor receptor in human breast cancer: correlation with cytosolic and nuclear ER receptors and with biological and histological tumor characteristics.

Epidermal growth factor receptor (EGFr) and cytosolic (cER) and nuclear (nER) estradiol receptors were quantified in 220 primary breast cancers. The EGFr was significantly more frequent (chi 2 = 5.9; P less than 0.025) and its concentration was significantly higher (P less than 0.001) among ER- tumors than in ER+ tumors. There was a significantly greater proportion (chi 2 = 6.4; P less than 0.05) of node involvement in EGFr+/ER+ tumors than in EFGr-/ER+. Increases in the proportion of EGFr+ in ER- tumors are parallel to Scarff-Bloom scores (chi 2 = 6.1; P less than 0.05) and there is a significant trend (Spearman rs = 0.25; P less than 0.05) towards increased EGFr concentrations with histologic dedifferentiation. In ER+ tumors the median concentrations of EGFr in the different age groups show a linear correlation (LCC = 0.89; P less than 0.05) and follow a parallel profile with the medians of nER. These findings support the hypothesis that EGFr is a bad prognosis factor and suggest that EGFr expression and concentration in ER+ tumors might be considered an estrogenic action mediated through the binding of ER to their nuclear acceptors.

[Vasoactive intestinal peptide receptor in a human pancreatic carcinoma cell line].

Vasoactive intestinal peptide (VIP) receptors were identified in a human pancreatic carcinoma cell line by radioreceptor assay, including time course, dissociation study, competitive inhibition, and cross reactions with secretin and glucagon, both of which are hormones of the same family. Peak binding of 125I-VIP to the cells occurred at 20-30 min at 37 degrees C. Displacement curve showed an increasing inhibition of binding with increasing concentration of unlabeled VIP(inhibited by 95% at 1 microM of VIP). KD of VIP receptors was 1.68 x 10(-10)M, and the number of binding sites was 3.6 x 10(5)/cell. It was also shown that VIP was able to induce cAMP production in this cell line, indicating that the VIP receptors in this cell line were biologically active.

Immunohistological study of oestrogen receptors in breast carcinomas that are biochemically receptor negative.

The reliability of an immunohistological method, applied to paraffin wax sections, was assessed for determination of oestrogen receptor content of biochemically oestrogen receptor negative breast carcinomata. Sixty consecutive tumours with oestrogen receptor concentrations of less than 10 fmol/mg cytosol protein, as estimated by dextran-coated charcoal biochemical assay, were examined. Paraffin wax sections were treated with DNAse before applying a peroxidase-anti-peroxidase method using ER-ICA monoclonal antibodies. Fifty one cases (85%) were negative, six (10%) weakly positive, and three (5%) were moderately positive. No strongly positive cases were seen. It is suggested that cases with weakly positive staining, especially when localised to a small area, should be regarded as negative. On the other hand, as the three moderately stained cases included two small tubular carcinomas and an invasive ductal carcinoma with high progesterone receptor concentrations, it is more likely that the biochemical assay in these cases represented false negative results due to sampling error or inclusion of fibrous or other non-neoplastic tissue in the assayed samples. It is concluded that the immunohistological method used here is fairly reliable and would be especially valuable for determination of oestrogen receptor content in small, mammographically detected tumours from which no tissue would be available for biochemical assay or frozen section examination.

Nuclear DNA content, histological grade, and clinical course in patients with nonpalpable mammographically detected breast adenocarcinomas.

Sixty-five consecutive female patients, age 35-83, with nonpalpable breast carcinomas detected by mammography were classified with both morphological and cytochemical malignancy grading. Cytochemically, the tumors were divided into euploid and aneuploid types indicating low and high malignancy potential, respectively. The mean follow-up time in the euploid group was 6.7 years and in the aneuploid group 8.0 years. No significant difference in mortality was observed in the two groups comprising 65% euploid and 35% aneuploid tumors. Our results here indicate that an early detection of breast cancer at a clinically occult and nonpalpable level leads to better prognosis even in patients with aneuploid tumors whose tumors otherwise are considered to be highly malignant.

Anti-estradiol immunoperoxidase labeling of nuclei, not cytoplasm, in paraffin sections, determines estrogen receptor status of breast cancer.

We evaluated a commercially available polyclonal antibody to 17 beta-estradiol as the basis for an estrogen receptor (ER) assay of breast carcinoma in formalin-fixed paraffin tissues and then compared it with both the ER-ICA antibody in serial paraffin sections and the biochemical assay of corresponding fresh tissue. Using the estradiol antibody, 49 of 50 cases showed some cytoplasmic staining; 38 cases had nuclear staining. Sensitivity and specificity for different proportions of positive nuclear and cytoplasmic staining were calculated using receiver-operator characteristic curves. The optimum correlation with the biochemical assay was obtained with nuclear staining alone. Greater than 30% nuclear positivity as a cut-off point yielded a sensitivity of 76% and a specificity of 82%. The corresponding ER-ICA values in 38 cases yielded a sensitivity of 93% and a specificity of 56%. The methodology for the ER-ICA assay was more technically demanding in paraffin sections than that of the estradiol antibody and considerably more expensive. This study is the first to show that with nuclear staining only, and not cytoplasmic staining, as the parameter of positivity, the immunocytochemical assay of ER with anti-17 beta-estradiol antibody in routinely processed, formalin-fixed, archival material is an accurate and specific method for the determination of the ER status of breast carcinoma.

[Nuclear morphometry and estrogen receptors in infiltrating ductal carcinoma of the breast]. / Morfometria nucleare e recettori estrogenici (RE) nel carcinoma duttale infiltrante della mammella.

In this study ten cases of breast infiltrating ductal carcinoma have been considered. In all of them the content of ER has been evaluated by using monoclonal antibodies. Five of them were ER positive and five were ER negative. For the morphometric study ten nuclei of each case have been considered. By using the S.A.M. (Shape Analytical Morphometry) work-station an analytical study of the nuclear shape was performed. The first step was the extraction of fundamental shape which describes the basic shape of original contour without its irregularities. It was obtained by using two parametric equations. The second step was the evaluation of shape asymmetry by S.A.E. (Shape Asymmetry Evaluator). Finally the contour irregularities were evaluated by Fourier analysis. Along with analytical parameters, dimensions (area, perimeter and maximum diameter) were considered too. All obtained data were submitted to univariate statistical analysis (Student's T test) to compare the two groups (ER positive and ER negative tumors). Area, perimeter and maximum diameter were significatively greater in ER negative cases while analytical parameters were not discriminant between the two groups.

Tenascin distribution in the normal human breast is altered during the menstrual cycle and in carcinoma.

Tenascin is a novel extracellular matrix glycoprotein which appears to have a major role in tissue development. Previous studies have stated that tenascin is absent from the normal human, rat and mouse breast, its distribution being restricted to embryonic and malignant mammary tissues. No previous studies have investigated tenascin distribution as a function of the normal menstrual cycle. Therefore this study addresses the cyclical appearance of tenascin in the normal breast and associated changes in distribution in preinvasive cancer (carcinoma-in-situ) and invasive infiltrating ductal carcinoma. Tenascin is present in the normal human adult mammary gland, principally in the basement membrane, sub-basement-membrane zone and delimiting layer of fibroblasts around the ductules. Both the distribution and quantity of tenascin change during the menstrual cycle. In carcinoma-in-situ (preinvasive cancer) tenascin is present in the attenuated basement membrane/sub-basement-membrane zone around the expanded ductules and in small amounts in the stroma. In infiltrating ductal carcinoma, tenascin is absent from the remnants of the basement membrane and sub-basement-membrane zone but greatly increased in the adjacent intralobular and interlobular stroma. Therefore, if tenascin is used as a basement membrane/sub-basement-membrane marker for distinguishing carcinoma-in-situ from invasive ductal carcinoma, the time of the menstrual cycle is of importance in interpreting the biopsy appearance. This study suggests that the optimal time for biopsy is between weeks 3 and 4 of the cycle, to avoid confusion between the normal low levels of tenascin (due to hormonal status) and those due to microinvasive disease.(ABSTRACT TRUNCATED AT 250 WORDS)

HER-2/neu oncogene expression and DNA ploidy analysis in breast cancer.

This study was performed to evaluate the correlation between HER-2/neu gene expression and DNA ploidy patterns. Forty-five cases of breast-cancer were analyzed. Immunohistochemical staining of HER-2/neu protein on frozen sections was used to detect the HER-2/neu protein, and the Feulgen DNA staining method was used to assess DNA amounts in the same tumor cells. Positive HER-2/neu overexpression was evaluated visually, and quantitation of the HER-2/neu protein was measured by image analysis. Twenty-two of the 45 cases were visually scored to be positive for the overexpression of the HER-2/neu protein, and these cases also contained above 10% HER-2/neu protein compared with a standard control cell line. All 22 of these cases had near-tetraploid DNA content. In contrast, cells, derived from the 23 cases that did not overexpress the HER-2/neu protein, contained DNA amounts that ranged from euploid (diploid) to varying degrees of aneuploid. The results of this study indicated that tumors that overexpress the HER-2/neu protein have tetraploid or near-tetraploid DNA content. This pattern could relate to the biological behavior of these tumors.