Which breast carcinomas need HER-2/neu gene study after immunohistochemical analysis? Results of combined use of antibodies against different c-erbB2 protein domains.

AIMS: Evaluation of HER2 gene amplification in breast cancers is a compelling, routine procedure. The aim of this work was to evaluate which breast carcinomas would really benefit from HER-2/neu gene analysis. METHODS AND RESULTS: We studied 130 invasive breast carcinomas by immunohistochemistry (IHC) using CB11 and TAB250 MAbs directed against different domains of the c-erbB2 molecule. From this series, we selected 106 cases (32 G1, 36 G2, and 38 G3) in which HER-2/neu gene analysis, using chromogenic in-situ hybridization (CISH), was successful. IHC results were scored using the FDA approved system with three score values: 0/1+ (negative), 2+, 3+ (positive). In addition, we developed a double scoring system with six score values (0/1+ 2+ negative, 3+, 4+, 5+, 6+ positive) obtained by summating the individual scoring values obtained with each MAb. All double scoring negative cases were non-amplified (100% sensitivity), whereas all cases scored 6+ were amplified. Double scoring values and CISH results were then correlated with grade and histological type. G1 ductal carcinomas and carcinomas of lobular and of special histological type did not show HER-2/neu amplification even in the presence of protein over-expression. CONCLUSIONS: The combined results of IHC analysis (double scoring values) obtained using MAbs directed against different c-erbB2 domains correctly indicates the HER-2/neu gene status in 57.5% of cases. In addition, simple morphological features such as low grade and special histological type are good predictors of the non-amplification of the HER-2/neu gene in breast carcinoma.

Expression of apocrine differentiation markers in neuroendocrine breast carcinomas of aged women.

Neuroendocrine (NE) breast carcinomas are a rare entity in young women; however, their frequency increases in aged patients. The present work demonstrates that NE breast carcinomas in elderly women can also express an apocrine immunophenotype and analyzes the histological and clinical aspects of such differentiation. A selected series of 50 NE tumors (positive for NE markers in >/=50% of the cells) was tested for the immunocytochemical expression of gross cystic disease fluid protein-15 (GCDFP-15). The results demonstrated that about 50% of moderately (G2) and well-differentiated (G1) NE breast carcinomas (mucinous, solid papillary, and solid cohesive histotypes) coexpressed the apocrine marker. In these cases, specific mRNA for GCDFP-15 (PIP) and for chromogranin A (ChA) was demonstrated using in situ hybridization (ISH). Carcinomas of the alveolar subtype (G2) and poorly differentiated carcinomas (G3), including one case of atypical carcinoid, were pure NE carcinomas, devoid of apocrine differentiation. The steroid receptor status of these lesions was evaluated to test a possible involvement of androgen receptors in apocrine differentiation. We demonstrated that the level of AR and the mean age of patients at diagnosis were significantly higher in apocrine than in nonapocrine differentiated tumors. The histological grade and the expression of estrogen receptor (ER) significantly influenced the prognosis of these NE carcinomas, either pure or NE-apocrine differentiated. The most original result of our study is therefore the demonstration of a possible divergent apocrine differentiation of NE breast carcinomas that might be regulated by the activation of androgen receptors in elder patients. In addition, the possibility for using Chs or GCDFP-15 serum values in the follow-up of these patients, as demonstrated in two cases of the present series, can justify the immunophenotyping of the tumors.

Extensive DNA fragmentation in oxyphilic cell lesions of the thyroid.

The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown.

Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry.

AIM: Antigen retrieval (AR) procedures are based on the effect of heating (by either microwave or pressure cooking treatments) on routinely fixed and paraffin embedded tissues. We observed that AR procedures restore the reactivity of endogenous biotin (EB) and report on the distribution of EB following AR in a series of routinely fixed and embedded tissues. METHODS AND RESULTS: Following pressure cooking or microwave treatments, a simple streptavidin-peroxidase staining revealed retrieved endogenous biotin (REB) in normal tissues (such as liver, kidney and adrenal cortex), in oxyphylic cells and in some tumours, especially in carcinomas of the kidney and of the adrenal cortex. In formalin-fixed (but not in alcohol-fixed) tissue sections, the heating procedures caused an intense and finely granular cytoplasmic reaction, following a routine streptavidin-conjugated peroxidase treatment. The staining was prevented by blocking of EB by a sequential avidin-biotin treatment. CONCLUSIONS: Retrieval of EB reactivity can cause pitfalls in diagnostic immunohistochemistry but, alternatively, it might also constitute a useful and novel diagnostic marker.

Antibody-dependent cytotoxic activity on human cancer cells expressing UK 114 tumor membrane antigen.

The monoclonal antibody (P-3 mAb) and the rabbit immune sera (RIS) recognizing a 14 kDa perchloric acid soluble protein extracted from goat liver (UK 114), locate this antigen on the cell membrane of several human cancer cell lines. UK 114-positive cells (e.g. HT 29 and KATO VI cells) undergo antibody-dependent cytolysis bl vitro. In nu/nu mice bearing xenografted HT 29 cells, tumor growth was markedly impaired by peri-tumoral injection of anti-UK 114 antibodies. These experiments suggest that human tumors expressing UK 114 over the cell membrane may undergo antibody-mediated cytolysis and growth control.

Immunophenotypic properties and estrogen dependency of budding cell structures in the developing mouse mammary gland.

The initial phase of growth of the parenchymal component of the mouse mammary gland is ductal clongation, which is mainly accomplished by proliferating cells in a specialized structure termed end bud. End buds are composed of multiple layers of epithelial cells (so called body cells) which are capped by a single layer of morphologically unique cells termed cap cells. We sought to examine the interrelationship between cap cells and other epithelial cell subclasses using a variety of antibodies to different keratin proteins and also antibodies to vimentin, actin and collagen IV. An extensive immunohistochemical characterization of the epithelial components of the developing and differentiating mammary gland demonstrated that cap cells were devoid of any immunohistochemically-detectable keratins but were positive for collagen IV. In contrast, the majority of cells in the end bud along with the luminal epithelial and myoepithelial cells were keratin positive. The body cells of the end bud were the only cells which were positive for antibody to keratin 6, a keratin which previously has been reported to be expressed in proliferating mammary epithelial cells. In addition, estrogen receptor was localized only to epithelial cells of ducts, alvcoli and body cells of end buds, but not to cap cells or myoepithelial cells. We interpret these results to suggest that cap cells are not totpotent stem cells but rather cells specialized in paving the way for ductal elongation as well as serving as precursors to myoepithelial cells.

Immunohistochemical detection of carcinoembryonic antigen (CEA) in non-neoplastic lung disease.

Carcinoembryonic antigen (CEA), though typically associated with malignant epithelial neoplasms, is known to be present at elevated levels even in the serum of normal individuals and of patients suffering from interstitial diseases of the lung. Few reports have addressed the question of the possible source of CEA immunoreactivity within the lung parenchyma. Two patients with elevated CEA serum levels were studied by immunohistochemistry on open lung biopsy specimens. Two different antibodies (one absorbed with non-specific cross-reacting antigen, NCA) were used. The results show that bronchiolar cells and type II pneumocytes are focally positive with both antibodies; the immunoreaction is preserved even after absorption with NCA. In agreement with experimental data on CEA synthesis in fetal bronchial cell lines, these findings indicate that interstitial lung disorders may induce abnormal CEA-like substance expression. In these cases, where no epithelial neoplasms subsequently develop, the cutoff level for CEA in serum should be raised. Bronchiolar and alveolar cells appear primarily responsible for CEA-like substance production.

Tumor types derived from epithelial and myoepithelial cell lines of R3230AC rat mammary carcinoma.

Epithelial and myoepithelial cells coexist in the rat R3230AC mammary tumor. To test the hypothesis that these two cell types constitute interactive but independent neoplastic populations, we obtained in vitro cell lines with epithelial or myoepithelial patterns and transplanted them in syngeneic animals. One stabilized line (EPI) and four cloned lines (A, C, D, E) with epithelial characteristics, confirmed by positive reactions for keratins in immunocytochemical and immunoblot tests, constantly gave rise in vivo to carcinomas, which, however, lacked structural and functional patterns typical of the original tumor. A fusiform shape and immunocytochemical characteristics of myoepithelial cells were observed in three clones (H, I, L), which in vivo gave rise to sarcomatous and mixed carcinosarcomatous neoplasms. These data are consistent with the above hypothesis and indicate that breast carcinomas derive from epithelial cells, while sarcomatous and carcinosarcomatous neoplasms can originate from myoepithelial cell proliferation. This study provides data suggesting myoepithelial cell involvement in the development of pathological entities occurring in the human breast and displaying mixed epithelial and stromal neoplastic components, i.e., cystosarcoma phylloides and sarcomatous metaplasia in carcinomas.

Cytological analysis of benign breast disease.

The different histological lesions of benign breast disease (BBD) are the result of interactions and regional prevalence of epithelial, myoepithelial, apocrine, and "null" (undifferentiated) cell types. We conducted an immunocytochemical analysis on 14 cases of BBD. Specific markers were employed to identify the different cell types; proliferation in these cells was revealed either by bromo-deoxyuridine uptake or PCNA ("cyclin") localization. The results indicate that apocrine cells do not undergo proliferation, representing therefore a terminally differentiated cell. The question related to the preneoplastic potential of BBD and on which cell type might possibly represent the precursor of in situ cancerous lesions remains unanswered. However, our data tend to exclude that such a putative proliferating precursor might be represented by apocrine or myoepithelial cells or even by the epithelial cells featuring epitheliosis.

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.

Immunocytochemical identification of proliferating cell types in mouse mammary gland.

To study cell proliferation in different cell types and segments of the mammary gland, we devised a dual staining procedure, combining nuclear labeling by 5-bromo-2'-deoxy-uridine (BrdU) uptake (revealed by a dark-brown precipitate) and an alternative (red or blue) cytoplasmic labeling by antibodies specific for the differentiation proteins of epithelial, myoepithelial, and secretory cell types. The following markers, revealed by APAAP or beta-galactosidase procedure, were selected: alpha-smooth muscle actin for the myoepithelial cells, keratin (detected by AE1 monoclonal) for the luminal epithelial cells, alpha-lactalbumin and beta-casein for the secretory cells. To follow the full process of organogenesis, the study was conducted in mouse mammary glands from virgin, primed, and lactating animals and from glands cultured in vitro under specific hormone stimulation. Cell proliferation was localized mainly in focal areas (end buds), and mostly corresponded to "null" undifferentiated cells. Estrogen and progestin stimulation induced a relative increase of proliferating differentiated cells of either epithelial or myoepithelial type, localized in ducts and alveolar structures. Prolactin stimulation induced proliferation in secretory cells.

Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.

alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.

Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody.

The myoepithelial cells of the sweat, mammary, tracheobronchial, and salivary glands are specifically identified by monoclonal antibody alpha-SM-1, which recognizes alpha smooth muscle actin and not the other actin isoforms. Basal or "reserve" cells in the stratified epithelia and excretory ducts of the salivary glands are negative, as well as all other epithelial cells in various organs. The reaction can be performed in routinely fixed and embedded tissues and is of practical interest in diagnostic histopathology. In immunoelectron cytochemistry, alpha-SM-1 antibody binds to the microfilament bundles in myoepithelial cells of the breast, but does not stain luminal cells and occasional basally located epithelial cells. These basal cells are morphologically and immunocytochemically distinct from the myoepithelial cells, and their nature and significance remain to be clarified.

Endocrine markers in argyrophilic carcinomas of the breast.

Argyrophilia in breast carcinomas is of uncertain significance. We tested a series of 20 cases of Grimelius-positive carcinomas with immunocytochemical markers of endocrine or exocrine differentiation. Fifty per cent of these tumors were positive, in a variable percentage of the neoplastic cells, with monoclonal antibodies against chromogranin, a specific marker of neuroendocrine differentiation. All cases were positive for neuron-specific enolase, but the significance and specificity of the reaction remain doubtful. The apparent positivity for alpha-lactalbumin, as found also by Clayton and coworkers, was found to be related to a contaminant, which is in fact also an endocrine marker. As with other types of breast carcinoma, all our cases were positive for epithelial membrane antigen, evidence that argyrophilic breast carcinomas, and specifically the chromogranin-positive subgroup, should be interpreted as endocrine neoplasms displaying multidirectional differentiation.