Usefulness of immunocytochemistry using a Breast Marker antibody cocktail targeting P63/cytokeratin7/18/cytokeratin5/14 for fine needle aspiration of the breast: a retrospective cohort study of 139 cases.

OBJECTIVE: The Breast Marker Cocktail from Biocare Medical comprises five antibodies recognising p63, and cytokeratins (CKs) 7, 18, 5 and 14. Immunohistochemistry using this cocktail is useful for diagnosing proliferative intraductal breast lesions. However, cytology using the cocktail has not been reported. METHODS: We report 139 cases of mammary samples collected by fine needle aspiration (FNA) for which histological diagnoses were available. After cell transfer, immunocytochemistry was performed using the cocktail, and clusters of cells were classified. A cluster with no or limited CK5/14 expression (<20% of cells) was classified as a monotonous cluster. One with more than 20% of cells showing CK5/14 expression was defined as a mosaic cluster. When at least one p63-positive cell was present, we defined it as a cluster with p63. We also evaluated background p63-positive myoepithelial cell densities. RESULTS: The diagnostic sensitivity and specificity for carcinomas were 97.8% (89/91) and 91.7% (11/12), respectively, using the criterion of two or more monotonous clusters lacking p63. Two false-negative cases were triple-negative cancers; one false-positive was an apocrine papilloma. The numbers of monotonous clusters with p63 differed significantly between benign lesions, ductal carcinoma in situ (DCIS)/lobular carcinoma in situ (LCIS) and invasive carcinomas (P < 0.001). The background myoepithelial cell density was significantly higher in fibroepithelial tumours than in other lesions (P < 0.001). CONCLUSIONS: Immunocytochemistry using this antibody cocktail showed good sensitivity and specificity for diagnosing breast cancers. Thus, this method is useful for mammary cytology using FNA.

Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes.

The intermediate filament cytoskeleton is essential for the development and maintenance of normal tissue function. A number of diverse recent observations implicate these filament systems in sensing stress and protecting cells against its worst consequences. Cells expressing severely disruptive keratin mutations, characteristic of Dowling-Meara EBS, were previously reported to show elevated responses to physiological stress, and partial disassembly of cell junctions was reported upon direct mechanical stress to the cells. Gene expression microarray analysis has therefore been used here to examine the broad spectrum of effects of mutant keratins. Many genes associated with keratins and other components of the cytoskeleton showed altered expression levels; in particular, many cell junction components are down-regulated in EBS cells. That this is due to the expression of the mutant keratins, and not to other genetic variables, is supported by observation of the same effects in isogenic cells generated from wild type keratinocytes transfected with the same keratin mutations in the helix boundary motifs of K14 or K5. Whilst the mechanism underlying this is unclear, these findings may help to explain other aspects of EBS-associated pathology, such as faster scratch wound migration, or acantholysis (cell-cell separation) in patients' skin. Constitutive stress combined with constitutively weakened cell junctions may also contribute to a recently reported increased risk of non-melanoma skin cancer in EBS patients.

Self-organization of keratin intermediate filaments into cross-linked networks.

Keratins, the largest subgroup of intermediate filament (IF) proteins, form a network of 10-nm filaments built from type I/II heterodimers in epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical stress. Like filamentous actin, keratin IFs must be cross-linked in vitro to achieve the high level of mechanical resilience characteristic of live cells. Keratins 5 and 14 (K5 and K14), the main pairing occurring in the basal progenitor layer of epidermis and related epithelia, can readily self-organize into large filament bundles in vitro and in vivo. Here, we show that filament self-organization is mediated by multivalent interactions involving distinct regions in K5 and K14 proteins. Self-organization is determined independently of polymerization into 10-nm filaments, but involves specific type I-type II keratin complementarity. We propose that self-organization is a key determinant of the structural support function of keratin IFs in vivo.

Proteomic assessment of sulfur mustard-induced protein adducts and other protein modifications in human epidermal keratinocytes.

Although some toxicological mechanisms of sulfur mustard (HD) have been uncovered, new knowledge will allow for advanced insight in the pathways that lead towards epidermal-dermal separation in skin. In the present investigation, we aimed to survey events that occur at the protein level in human epidermal keratinocytes (HEK) during 24 h after exposure to HD. By using radiolabeled (14)C-HD, it was found that proteins in cultured HEK are significant targets for alkylation by HD. HD-adducted proteins were visualized by two-dimensional gel electrophoresis and analyzed by mass spectrometry. Several type I and II cytokeratins, actin, stratifin (14-3-3sigma) and galectin-7 were identified. These proteins are involved in the maintenance of the cellular cytoskeleton. Their alkylation may cause changes in the cellular architecture and, in direct line with that, be determinative for the onset of vesication. Furthermore, differential proteomic analysis was applied to search for novel features of the cellular response to HD. Partial breakdown of type I cytokeratins K14, K16 and K17 as well as the emergence of new charge variants of the proteins heat shock protein 27 and ribosomal protein P0 were observed. Studies with caspase inhibitors showed that caspase-6 is probably responsible for the breakdown of type I cytokeratins in HEK. The significance of the results is discussed in terms of toxicological relevance and possible clues for therapeutic intervention.