Patrizia Gugliotta

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2‐amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual‐colour FISH, were studied by microarray‐based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling‐path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray‐based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted. Copyright

Nonspecific staining of mast cells by avidin-biotin-peroxidase complexes (ABC).

A nonspecific staining of mast cells by avidin-biotinylated peroxidase complexes (ABC) has been observed and related to an ionic binding of the basic residues of avidin (isoelectric point at pH 10) and peroxidase to the sulphate groups of heparin. This affects the correct interpretation of the results of the immuno-peroxidase ABC procedure, especially in mast cells-rich areas such as the lymphoid tissues. The spurious staining can be prevented by using the ABC solution at pH 9.4 (instead of pH 7.6 as usually recommended): this high pH does not affect the previous binding of primary antibodies nor the affinity of avidin to biotin. The modified ABC procedure provides a clear background and a sharp specific staining and can be recommended for routine use.

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.

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

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.

Immunohistochemical analysis of benign and malignant papillary lesions of the breast.

The histocytological diagnostic criteria and recently developed immunohistochemical procedures selective for either the epithelial or the myoepithelial mammary cells have been tested in a series of 60 cases of papillary lesions of the breast. These included 15 benign solitary intraductal papillomas, 41 papillary carcinomas (29 pure and 12 associated with other types of in situ or invasive ductal carcinoma), and four cases of “suspected” papillary carcinomas.Markers for epithelial cells (EMA) and for apocrine metaplasia (GCDFP-15) did not permit a distinction between benign and malignant papillary lesions; however immunocytochemical staining for CEA using monoclonal antibodies, and for actin (a marker of the myoepithelial cells) was discriminative in this respect.Benign papillomas have a basal layer of actin-rich myoepithelial cells; the cytoplasm of the epithelial cells is CEA negative. Papillary carcinomas lack the myoepithelial layer, except in areas where multiple papillomas are present, associated with ductal or papillary cancer CEA was detected in 85% of carcinomas. Two of the cases of “suspected carcinoma” lacked myoepithelial cells and were interpreted as carcinomas.It is concluded that the immunocytochemical methods for cell markers can offer valuable data in the study and diagnosis of papillary lesions of the breast; it is difficult however, to be categorical in borderline cases since in our experience, the behavior of the malignant papillary lesions of the breast is usually favorable. Residual foci of multiple intraductal papillomas were found in seven cases of papillary carcinoma, supporting the pre-neoplastic potential of this condition.

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 alphalactalbumin, as found also by Clayton and coworkers (8), 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.

Actin-Rich (myoepithelial) cells in ductal carcinoma-in-situ of the breast

SummaryThe distribution of the myoepithelial cells in 32 cases of ductal carcinoma-in-situ (DCIS) of the breast (11 not associated, 21 associated with invasive carcinoma) was investigated with a recently developed immunoperoxidase method for actin.Actin-rich myoepithelial cells were detected at the periphery of some ducts, however, their presence was neither constant nor continuous. Large areas of DCIS were devoid of a myoepithelial cell layer and the neoplastic cells were directly in contact with the stroma. No differences related to the histological pattern of DCIS or the presence and absence of invasive carcinoma were noted.The behaviour of the myoepithelial cells in ductal carcinoma appears different from that observed in cases of lobular carcinoma (Bussolati 1980) and of cystic disease, and may thus be of diagnostic interest.The selective destruction of myoepithelial cells in cases of DCIS might result in a focal disruption of the basement membrane, thus facilitating invasion.

Benign clear cell (‘Sugar’) tumor of the lung A light microscopic, histochemical, and ultrastructural study with a review of the literature

A case of a benign clear cell “sugar” tumor of the lung is reported. Light microscopy showed a uniform proliferation of clear cells filled with abundant glycogen. At the ultrastructural level, tumor cells were rich in free monogranular and rosette‐forming glycogen, but no membrane‐bound glycogen was demonstrated. Some cells showed plasma membrane interdigitations, microvilli, and macula occludens‐type junctions. Many polymorphic secretory and sporadic haloed neurosecretory‐like granules were observed, but argyrophil stains as well as a large set of immunohistochemical reactions specific for APUD derivation had negative results. A literature review of this puzzling entity with particular emphasis on the histogenetic hypotheses is presented, and a derivation from epithelial nonciliated bronchiolar (Clara) cells or epithelial serous cells is suggested.

Association of breast carcinoma and multiple intraductal papillomas: an histological and immunohistochemical investigation

To identify histological preneoplastic lesions and early neoplastic foci, a histological and immunohistochemical study has been conducted on a series of 18 cases with the rare association of multiple intraductal papillomas and in situ breast carcinoma. The pathological and clinical data of these cases have been collated.

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.