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.

Application of multispectral imaging in quantitative immunohistochemistry study of breast cancer: a comparative study.

Multispectral imaging (MSI) based on imaging and spectroscopy, as relatively novel to the field of histopathology, has been used in biomedical multidisciplinary researches. We analyzed and compared the utility of multispectral (MS) versus conventional red-green-blue (RGB) images for immunohistochemistry (IHC) staining to explore the advantages of MSI in clinical-pathological diagnosis. The MS images acquired of IHC-stained membranous marker human epidermal growth factor receptor 2 (HER2), cytoplasmic marker cytokeratin5/6 (CK5/6), and nuclear marker estrogen receptor (ER) have higher resolution, stronger contrast, and more accurate segmentation than the RGB images. The total signal optical density (OD) values for each biomarker were higher in MS images than in RGB images (all P < 0.05). Moreover, receiver operator characteristic (ROC) analysis revealed that a greater area under the curve (AUC), higher sensitivity, and specificity in evaluation of HER2 gene were achieved by MS images (AUC = 0.91, 89.1 %, 83.2 %) than RGB images (AUC = 0.87, 84.5, and 81.8 %). There was no significant difference between quantitative results of RGB images and clinico-pathological characteristics (P > 0.05). However, by quantifying MS images, the total signal OD values of HER2 positive expression were correlated with lymph node status and histological grades (P = 0.02 and 0.04). Additionally, the consistency test results indicated the inter-observer agreement was more robust in MS images for HER2 (inter-class correlation coefficient (ICC) = 0.95, r s = 0.94), CK5/6 (ICC = 0.90, r s = 0.88), and ER (ICC = 0.94, r s = 0.94) (all P < 0.001) than that in RGB images for HER2 (ICC = 0.91, r s = 0.89), CK5/6 (ICC = 0.85, r s = 0.84), and ER (ICC = 0.90, r s = 0.89) (all P < 0.001). Our results suggest that the application of MS images in quantitative IHC analysis could obtain higher accuracy, reliability, and more information of protein expression in relation to clinico-pathological characteristics versus conventional RGB images. It may become an optimal IHC digital imaging system used in quantitative pathology.

Immunohistochemical application of D2-40 as basal cell marker in evaluating atypical small acinar proliferation of initial routine prostatic needle biopsy materials.

D2-40 has been recently discovered as a lymphatic endothelial cell marker, and some investigators have found that D2-40 is also expressed in myoepithelial cells of salivary gland or breast. In this study, we evaluated D2-40 expression of basal cells and applied D2-40 immunohistochemistry in the combination of P504S, cytokeratin 5, and p63 for ten lesions with atypical small acinar proliferation (ASAP) in initial prostatic needle biopsy. As a result, D2-40 was expressed in basal cells, lymphatic endothelial cells, and some stromal fibroblasts of normal prostatic tissue. Among ten ASAP lesions, the final diagnosis of seven lesions was resolved by combination immunohistochemistry. D2-40 was comparable to cytokeratin 5 and p63 as a basal cell marker, and there were no lesions that failed to provide an accurate final diagnosis using only D2-40 immunohistochemistry without cytokeratin 5 or p63. However, we found some D2-40-positive stromal fibroblasts or D2-40-positive lumen-collapsed lymphatic vessels neighboring atypical glands. Pathologists should pay attention to avoid recognizing these cells as basal cells. In conclusion, the combination of immunohistochemistry of P504S, cytokeratin 5, p63, and D2-40 may contribute to the accurate diagnosis of ASAP in the initial prostatic needle biopsy.

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.