Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice.

We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.

Overexpression of p53 is a late event in the development of malignant melanoma.

Overexpression of the p53 gene product has been observed in a high percentage of malignant melanomas. To evaluate the role of this protein in the development of melanoma, we examined p53 expression in benign, premalignant, and malignant melanocytic lesions. Using the antibodies DO-7 and 1801, which recognize both wild-type and most mutant forms of the p53 protein, we analyzed by immunohistochemical staining 26 benign nevi, 34 dysplastic nevi from patients at low risk for the development of melanoma, 22 dysplastic nevi from patients at high risk for the development of melanoma, 61 primary melanomas (including 15 that arose from dysplastic nevi), and 10 metastatic melanomas. Expression of the p53 protein was not observed in any of the benign or dysplastic nevi. Of the primary melanomas only 3 (5%) demonstrated nuclear staining, whereas 70% of the metastatic melanomas showed a positive reaction for p53. These data suggest that overexpression of the p53 gene product is a late event in the progression of melanoma and consequently indicate that expression of this protein cannot be used as a marker to identify patients at high risk for the subsequent development of melanoma.

MIB-1 proliferative activity is a significant prognostic factor in primary thick cutaneous melanomas.

Although the Breslow measurement of tumor thickness of melanoma is the most significant predictor of survival, the biologic behavior of thick lesions remains unpredictable. MIB-1, a monoclonal antibody to a Ki-67 epitope, recognizes all proliferating cells. Unlike Ki-67 antibody, which requires frozen tissue, MIB-1 can be used on formalin-fixed tissue. Proliferation, measured by MIB-1 expression and mitotic index, was assessed as a prognostic factor in a group of patients with clinical stage I thick cutaneous melanoma (tumor thickness 4 mm or greater), for which predicted survival is low. From a melanoma data base, 97 patients with this type of melanoma were identified. Of these, 64 had lesional tissue available for study. The median follow-up time was 3.8 years (range 0.42-13.6 years). The percentage of MIB-1 reactivity was scored as low at less than 10% (n = 33), intermediate at 10% to 20% (n = 17), and high at greater than 20% (n = 14). Melanomas with high MIB-1 reactivity were associated with significantly poorer cause-specific survival compared with tumors with intermediate (p < 0.0001) or low MIB-1 reactivity (p = 0.0025). Multivariate analysis demonstrated that MIB-1 reactivity was a significant independent prognostic factor related to cause-specific survival (p = 0.0002) and was more sensitive than tumor thickness or mitotic index in this select group of high-risk patients. Identification of individuals with stage I thick cutaneous melanoma who are at risk of recurrent disease may improve patient management as new therapeutic modalities become available.

An improved statistical approach: can it clarify the role of new prognostic factors for breast cancer?

Recently, there has been a proliferation of new biomarkers, some of which may lead to an improved prognostic index or may influence treatment selection. However, there are methodological and statistical issues that require attention in assessing the role and use of these prognostic factors. Between 1977 and 1986, 1097 primary breast cancer patients were accrued for multidisciplinary research at the Henrietta Banting Breast Centre, Women's College Hospital; follow-up to 1990 is complete for 96% of the patients. Data for these patients are used here to illustrate strategies: (1) for the comparison of results from diverse assessments of biomarkers; (2) for the improved comparability of inter-laboratory results; (3) for the examination of the results from monoclonal or polyclonal antibody assays for possible clinically relevant bimodality; (4) for good statistical resolution of overlapping distributions; (5) that involve the use of quantitative values for prognostic factors whenever possible; and (6) for improved multivariate analyses. Good data handling and analyses may enable more accurate and rapid assessment of new prognostic factors, thereby expediting and improving their clinical application.

Ultrastructural and immunohistochemical characteristics of mucoepidermoid carcinoma of the breast.

Mucoepidermoid carcinoma is a rare subtype of breast carcinoma. In an attempt to elucidate the histogenesis of these tumors, two mucoepidermoid carcinomas were studied electron microscopically and immunohistochemically with antisera to S-100 protein and epidermal cytokeratin. Light microscopic examination showed these tumors to be composed of mixtures of neoplastic mucus-secreting, squamous, and intermediate cells. Ultrastructural examination revealed squamous cells, mucus-secreting luminal cells, and classic myoepithelial cells. The intermediate cells, which contained focal peripheral myofilaments with dense bodies and pinocytotic vesicles associated with centrally aggregated tonofilaments, were interpreted as modified myoepithelial cells. Classic dendritic myoepithelial and intermediate (modified myoepithelial) cells stained with antisera to S-100 protein and epidermal cytokeratin. Both the ultrastructural and the immunohistochemical features indicate that myoepithelial and duct luminal cells play an important role in the histogenesis of mucoepidermoid tumors of the breast.

Tenascin differentiates dermatofibroma from dermatofibrosarcoma protuberans: comparison with CD34 and factor XIIIa.

Differentiation of dermatofibroma (DF) from dermatofibrosarcoma protuberans (DFSP) can be difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP. However, there is overlap and lack of specificity of their expression. Tenascin is an extracellular matrix glycoprotein that is involved in embryogenesis, carcinogenesis, and wound healing. The aim of the study was to assess the role of tenascin in DF and DFSP and compare the results with those obtained with CD34 and Factor XIIIa. Immunohistochemical staining was performed on 20 cases each of DFSP and DF, using antibodies to tenascin, CD34 and Factor XIIIa, and the streptavidin biotin technique. Positivity for all 3 antibodies was assessed within the tumors. Tenascin expression was also assessed at the dermal-epidermal junction. Strong tenascin positivity was noted at the dermal-epidermal junction overlying the lesion in 20 of 20 cases of DF (100%) and was negative over the lesion in 20 of 20 cases DFSP (100%). Tenascin was noted within the lesion of 80% of both DF and DFSP (16/20 cases). CD34 was strongly expressed in 16 of 20 (80%) DFSP and 5 of 20 (25%) DF, whereas Factor XIIIa was strongly expressed in 19 of 20 (95%) DF and 3 of 15 (15%) DFSP. Although CD34 was expressed in 80% DFSP and Factor XIIIa in 95% of DF, there was overlap in their expression in the 2 types of tumors. The increased expression of tenascin at the dermal-epidermal junction overlying the lesion in DF but not in DFSP, differentiated these 2 tumors. In contrast, tenascin expression within the lesion did not differentiate DF from DFSP.

Origin of the desmoplasia in desmoplastic malignant melanoma.

Four cases of desmoplastic malignant melanoma were examined light microscopically and immunohistochemically. Electron microscopy was performed in three cases. Light microscopy showed that all tumors were composed of neoplastic spindle cells that infiltrated between mature collagen bundles in the reticular dermis. Some of the spindle cells had bizarre nuclei, whereas other spindle cells resembled normal fibroblasts. Melanin could not be demonstrated in any of the tumors by histochemical techniques. Electron microscopic examination of the spindle cells showed prominence of rough endoplasmic reticulum, which was dilated and filled with flocculent material and occasional collagen fibrils. The same cells contained aggregates of non-membrane-bound melanin granules and pre-melanosomes. Some cells also showed features of myofibroblasts. Immunoperoxidase staining with anti-S100 protein antibody demonstrated positivity of the spindle cells as well as of melanocytes in the basal layer of the epidermis. Scar tissue and fibroblasts did not stain. These findings show that the desmoplastic component of these malignant melanomas derives from melanocytes that have undergone adaptive fibroplasia. Therefore, in assessing depth of invasion in a malignant melanoma, measurements should include the desmoplastic areas.

Role of antibody to S100 protein in diagnostic pathology.

Normal tissues and various tumors were examined for S100 protein, using anti-S100 protein antiserum, in an immunoperoxidase reaction. Among normal tissues, in addition to the previously reported presence of S100 protein in some neurons, glial, and Schwann cells of the nervous system, melanocytes and Langerhans cells of the skin, interdigitating reticulum cells of lymph nodes, and chondrocytes, we demonstrated it in myoepithelial cells and ducts of sweat glands, salivary glands, and the breast, serous glands of the lung, fetal neuroblasts, and sustentacular cells of the adrenal medulla. Among neoplasms, S100 protein previously has been reported in neurogenic tumors, melanomas, and neuroblastomas; we have demonstrated it in mixed sweat gland tumors, histiocytosis X, pleomorphic adenomas of the salivary gland, medullary carcinomas of the breast, bronchioloalveolar carcinomas of the lung, sustentacular cells of pheochromocytomas, teratomas of the ovary, and tumors of cartilage (enchondromas, osteochondromas, and chondrosarcomas). With S100 protein producing tumors, a normal progenitor cell was identified, indicating that demonstration of S100 protein in tumors confirmed their origin.

The use of cytoskeletal characteristics of tumor cells for the diagnosis of colon and breast adenocarcinomas.

The cytoskeleton of colon (11 cases) and breast adenocarcinomas (9 cases) was characterized with the use of immunohistochemistry on tissue sections and one- and two-dimensional (2-D) gel electrophoresis of cytoskeletal extracts of tumor cells. By immunofluorescence, antibodies to epidermal cytokeratin (CK) and Mallory body CK recognized cytoplasmic filaments +/- desmosomal contacts, respectively, in both colon and breast adenocarcinomas. In addition, cytoskeletal extracts of both tumors showed similar CK polypeptides by 2-D gel electrophoresis. By immunoperoxidase, anti-actin antibody stained the apical margin of tumor cells in eight (73%) colon adenocarcinomas and four of five metastases, while diffuse cytoplasmic staining was seen in only one (9%) breast adenocarcinoma and not in five metastases. With 2-D gel electrophoresis, a cytoskeletal-associated doublet polypeptide was found in seven (64%) colon adenocarcinomas but not in the breast adenocarcinomas. By immunoblotting, the doublet did not consist of CK polypeptides, vimentin, or type IV collagen. These findings may facilitate the differentiation of colon and breast adenocarcinomas.

Immunohistochemical localization of epidermal and Mallory body cytokeratin in undifferentiated epithelial tumors. Comparison with ultrastructural features.

Twenty-one anaplastic tumors were studied by light microscopy (LM), immunoperoxidase staining using anti-epidermal cytokeratin (ECK) and anti-Mallory body cytokeratin (MBCK) antibodies, and electron microscopy (EM), to determine whether an epithelial origin could be confirmed. The tumors were derived from lung, stomach, colon, breast, uterus, kidney, bladder, and mesothelium. By LM, the tumors consisted of either large and polygonal, spindle or small, round cells. With immunoperoxidase staining, 11 (52%) of the anaplastic tumors were positive for ECK, positivity being either absent or only weak in the main tumor mass, but marked in areas of infiltration and metastases. In contrast, all of the anaplastic tumors were positive for MBCK in the main tumor mass, infiltrating areas, and metastases. In the case of adenocarcinomas, staining was either web-like or diffuse throughout the cytoplasm with concentration occurring at the cell surface, whereas in mesotheliomas, the staining was either diffuse or showed focal perinuclear accentuation. Twelve of 13 anaplastic tumors examined by EM showed epithelial features (desmosomes, tonofilaments, lumina, and/or microvilli). As controls, 21 non-epithelial tumors (five melanomas, eight sarcomas, and eight lymphomas) showed no reactivity with either cytokeratin antibody. These studies show that the epithelial nature of undifferentiated and poorly differentiated tumors can be confirmed by immunohistochemistry using anti-cytokeratin antibodies.

Distinct keratin patterns demonstrated by immunoperoxidase staining of adenocarcinomas, carcinoids, and mesotheliomas using polyclonal and monoclonal anti-keratin antibodies.

The authors assessed whether distinct patterns for keratin could be demonstrated in 10 adenocarcinomas, 10 carcinoids, and 4 mesotheliomas by an immunoperoxidase reaction using 3 polyclonal and 3 monoclonal antibodies to keratin. When color development in diaminobenzidine (DAB) was allowed to proceed for less than 2 minutes, distinct patterns for keratin could be demonstrated using two polyclonal and two monoclonal antibodies; these were plasma membrane and/or web-like in the adenocarcinomas, punctate or crescentic in the carcinoids, and perinuclear in the mesotheliomas consisting of tumor cells with abundant cytoplasm. Immunoelectron microscopy using protein A colloidal gold confirmed these results. When color development in DAB was allowed to proceed for more than 2 minutes, only diffuse staining was seen. The distinct patterns of immunostaining for keratin obtained with the shorter color development were helpful in differentiating adenocarcinomas, carcinoids, and mesotheliomas.

An investigation of cut-points for primary breast cancer oestrogen and progesterone receptor assays.

Oestrogen and progesterone receptor (ER and PgR) assay values are frequently used in medical decision-making for breast cancer patients. We have proposed statistical standardization of receptor assay values to improve inter-laboratory comparability, and now report the use of standardized log units (SLU) to investigate the effects of ER and PgR cut-points on time to first recurrence outside the breast (DFS). Between 1980 and 1986, there were 678 primary breast cancer patients treated at the Henrietta Banting Breast Centre (HBBC). The effects of ER and PgR cut-points were examined with multivariate analyses considering the variables: age, tumour size, nodal status, weight and adjuvant treatment. We considered receptor assay cut-points ranging from - 1.0 to + 1.0 SLU (ER between 7 and 166 fmol/mg protein; PgR between 7 and 181 fmol/mg protein). PgR was included in the multivariate prognostic models more often than ER, although patients had a better prognosis with both larger ER and PgR values. There was no best cut-point for ER or PgR, and there was strong evidence that ER and PgR should be considered as continuous rather than dichotomous (negative, positive) variables. Patient prognosis should also be more comparable with SLU.

Alternative multivariate modelling for time to local recurrence for breast cancer patients receiving a lumpectomy alone.

Certain prognostic factors (patient and/or tumour characteristics) may be associated with low (or high) risk for local recurrence. Patients with these characteristics could be candidates for less (or more) adjuvant therapy or a less (or more) aggressive surgical approach. However, the assessment of many factors can be problematic with the standard multivariate technique-a Cox proportional hazards model and step-wise regression. We compared the results obtained when using a Cox model with those from four alternative models (exponential, Weibull, log logistic and log Normal) in step-wise and all subset regressions. Between 1977 and 1986, 293 primary invasive breast cancer patients were treated at the Henrietta Banting Breast Centre with a lumpectomy with or without an axillary dissection, and with no postoperative adjuvant therapy. The variables considered were age, lymph node status, tumour size, estrogen receptor (ER), progesterone receptor (PgR), histologic grade, nuclear grade, carcinoma in situ (CIS), amount of CIS, and presence of tumour emboli. With follow-up to 1991, nodal status was not found to be included in the step-wise Cox model, although it was in the step-wise exponential, Weibull and log Normal models, and in the best all subset models for all model types. The variables tumour emboli, ER, age, CIS and nodal status were consistently included in the best all subset regressions, regardless of model type. In the 1993 follow-up, the variables in the step-wise Cox model were tumour emboli, ER, age, CIS and nodal status. The multivariate consideration of all possible subsets of regression variables led to an earlier indication of the importance of nodal status, while the data strongly supported accelerated failure time models over the Cox model.

Cystosarcoma phylloides of the breast and its mimics. An immunohistochemical and ultrastructural study.

Cystosarcoma phylloides of the breast is a tumor composed of breast ducts and a cellular stromal component that can be benign or malignant. The origin of the stromal cells is controversial. We undertook an immunohistochemical and ultrastructural study of 11 cases of cystosarcoma phylloides to assess the histogenesis of the stromal component. By light microscopy, 4 were diagnosed as benign, and 7 were diagnosed as malignant. Antibodies to vimentin, desmin, actin, high- and low-molecular-weight keratins, and S100 protein were used for immunohistochemical staining. In the 4 benign cases of cystosarcoma phylloides, the stromal cells stained positively only for vimentin. In the malignant tumors, the spindle cell component stained for vimentin in all the cases. In addition, the malignant stromal cells coexpressed desmin in two cases and keratin and S 100 protein in another case. By electron microscopy the stromal component in the benign case and in two of five malignant cases was composed of a mixture of fibroblasts and myofibroblasts. The entire neoplastic stroma in two other malignant cases showed features of smooth-muscle differentiation, whereas in another case all the stromal cells showed myoepithelial differentiation. Thus, in benign and malignant cystosarcoma phylloides, the stromal component consists of a mixture of fibroblasts and myofibroblasts. Leiomyosarcomas and myoepitheliomas can mimic malignant cystosarcoma phylloides, but immunohistochemistry and electron microscopy can differentiate these entities. This is important since their biologic behavior is different.

Epithelial proliferation in thymic hyperplasia. An immunohistochemical study and correlation with the developing fetal thymus.

Thymic hyperplasia is a B-cell lymphoid proliferation in which an epithelial component has not, to our knowledge, been previously described. We present a case of thymic hyperplasia in which numerous lymphoid follicles with germinal centers were partially surrounded by small sheets of spindle and epithelioid cells. Electron microscopy confirmed the epithelial nature of these cells. Immunostaining was performed using antibodies to keratins, S100 protein, and two B-cell markers, LN1 and MB2. The proliferated epithelium stained only for high-molecular-weight keratin, whereas the lymphoid tissue stained positively for both B-cell markers. To determine the origin of the proliferated epithelium, the staining was compared with that of the developing fetal and normal adult thymus. We have shown that during fetal development, the keratin composition of thymic epithelium changes from staining predominantly with low- to high-molecular-weight keratin. The immunostaining characteristics of the epithelium in this case of thymic hyperplasia suggest an origin from adult-type epithelium. Furthermore, the association of S100-positive interdigitating reticulum cells with the proliferated epithelium suggests that it is of medullary origin. Our results indicate that epithelial proliferation can be an important component of thymic hyperplasia.

Primary multilobulated B-cell lymphoma of the breast.

Malignant lymphomas with multilobulated nuclei are recently recognized neoplasms. We report a case of multilobulated B-cell lymphoma arising in the breast. The light microscopic, electron microscopic, and immunohistochemical features are described and compared with those of previously reported T- and B-cell multilobulated lymphomas from other sites. A follicular center cell origin of this lymphoma is postulated. To our knowledge, this is the first report of a primary multilobulated lymphoma of the breast.

Expression and amplification of neu oncogene in pleomorphic adenomas of salivary gland.

Amplification of the neu oncogene and overexpression of its product was found to be a potential marker for aggressive biological behavior in breast cancer. However, the expression of the neu oncoprotein in normal breast ducts and myoepithelial cells has also been demonstrated. Hence, we examined normal salivary gland tissue and 15 cases of pleomorphic adenoma for the expression of the neu oncoprotein by immunohistochemistry using two polyclonal antibodies and 12 cases for the amplification of the neu oncogene using slot blot hybridization. Immunohistochemistry in the normal salivary gland revealed positive staining of all ductal cells. In the pleomorphic adenomas all cellular elements stained to a variable degree. The positive staining was seen in the ductal cells, in the solid sheets, and in chondroid, myxoid, and metaplastic foci. The normal salivary gland and 11 of 12 cases of pleomorphic adenoma showed no increase in copy number of the neu oncogene, whereas one case showed threefold amplification. These results indicate that the neu oncoprotein is expressed but the neu copy number is not increased in normal salivary gland epithelium and in most pleomorphic adenomas. The threefold amplification of the gene in one case may indicate an aggressive biological behavior.

Myoepithelial cells in salivary gland tumors. An immunohistochemical study.

Normal salivary glands and 55 salivary gland tumors were examined by immunostaining (immunoperoxidase [IMP] and immunofluorescence [IMF]) to identify myoepithelial cells (MCs) and speculate on their role in the histogenesis of the tumors. The classic (C) MCs of normal salivary glands stained by IMP with antibodies to cytokeratin and S100 protein and stained by IMF with the same antibodies and with antibodies to vimentin and actin. Modified (M) MCs of pleomorphic adenomas stained positively by IMP and IMF with all of the preceding antibodies. In many mucoepidermoid carcinomas, adenoid cystic carcinomas, and basal cell adenomas, variable numbers of CMCs and MMCs stained positively by IMP with anti-cytokeratin and anti-S100 protein antibodies. No MCs were detected in adenolymphomas or acinic cell carcinomas. We believe that MCs play a major role in the histogenesis of pleomorphic adenomas and may also be important in many mucoepidermoid carcinomas, adenoid cystic carcinomas, and basal cell adenomas.

Controlling for variability in research on normal aging and neuropsychological populations discourse analysis and applications: studies in adult clinical populations.

There has been an increase in research during the past few years on discourse, with particular focus on normal older adults and clinical populations, e.g., aphasic and head-injured patients and patients with Alzheimer's disease. A recent edited book, Discourse Analysis and Applications: Studies in Adult Clinical Populations, reviews some of this work. Research on normal older adults and clinical populations raise a number of methodological problems, in particular, variability. Such issues are not easily resolved as we currently have no standard experimental designs for this type of research. This review seeks to point out some of these methodological issues that researchers face and provides some possible solutions.

Discourse analysis in neuropsychology: comment on Chapman and Ulatowska.

This paper discusses a recent article by Chapman and Ulatowska (1989, Brain and Language, 36, 651-658) on discourse analysis in aphasia. As such, the research in this area is interdisciplinary drawing from neuropsychology, as well as cognitive psychology, and, in part, aging. We illustrate problems that can arise when theoretical constructs and methodological considerations in this interdisciplinary approach are not rigorously observed. It is argued that definitive conclusions regarding the functional organization of the brain and discourse can be offered only when discourse analysis uses the state of the art from neuropsychology and cognitive psychology.