Cytokeratin expression in malignant melanoma: potential application of in-situ hybridization analysis of mRNA
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Occasional reports indicated cytokeratin (CK) protein expression (mainly by immunohistochemistry) in malignant melanoma (MM) and suggested an association with unfavorable clinical parameters. However, the mRNA expression of CK and its clinicopathologic significance in MM has not been specifically evaluated. We investigated the mRNA and protein expression of nine CKs in melanoma cell lines and tissues, in particular the prognostic significance of CK18 mRNA expression. Reverse transcription (RT)-PCR (CK6–10, 14 and 18–20), in-situ hybridization (ISH) (CK18), and western blotting (CK18 and pan-cytokeratin AE1/AE3) were performed on MM cell lines A375, A875, M14, and SK-MEL-1. Eighty MM tissue samples were analyzed by ISH and immunohistochemistry for CK18 expression. The mRNA of CK6–8, 10, 14, 18, and 19 (but not CK9 and 20) was detected in one to four of the melanoma cell lines by RT-PCR. CK18 was detected in all four cell lines by RT-PCR, ISH, and western blotting. CK18 mRNA ISH was positive in three of 30 (10.0%), 10 of 25 (40.0%), and 12 of 25 (48.0%) of primary cutaneous, primary mucosal, and metastatic melanomas, respectively (overall positivity: 25 of 80, 31.3%). CK18 immunostaining was only observed focally in eight of 80 (10.0%) of MM tissue samples, and AE1/AE3 immunostaining was altogether negative. Significantly, CK18 mRNA ISH positivity (but not protein immunohistochemistry) was associated with poorer prognosis by both univariate analysis (P<0.001) and multivariate analysis (relative risk=5.430, 95% confidence interval 2.246–13.128, P<0.001). CK18 mRNA could be identified in one-third of melanoma tissue samples and is an adverse prognostic factor. ISH is superior to immunohistochemistry for analyzing CK18 expression in MM.Keywords:
Immunostaining
A simple method for the harvest of bladder cell types from surgical specimens was used to generate strains of normal human urothelial cells that could be reproducibly cultivated, passaged and extensively expanded in serum-free medium. Immunostaining of the bladder epithelial cells with broadly reacting anti-cytokeratin antibodies and with an anti-cytokeratin antibody specific to cytokeratin 7, a transitional cell marker, indicated that they expressed a stable epithelial phenotype with serial passage. Low levels of immunostaining for E-cadherin and low levels of E-cadherin messenger ribonucleic acid, as determined by Northern blot analysis, and strongly positive immunostaining with an anti-vimentin antibody indicated collectively that the uroepithelial cells express a nonbarrier-forming phenotype under these culture conditions. However, when the urothelial cells were implanted subcutaneously into athymic mice on biodegradable synthetic polymers, they formed multilayered structures, suggesting that they retain the capability to differentiate in a living host. The urothelial cells proliferated in an epidermal growth factor independent manner and expressed high levels of transforming growth factor-alpha and amphiregulin messanger ribonucleic acids, suggesting the possibility of autocrine regulation of growth by epidermal growth factor-like factors. Cytogenetic analysis indicated that urothelial cells cultured for 6 passages possessed a normal chromosomal complement. These results demonstrate that primary cultures of autologous human bladder epithelial cells can be extensively expanded in vitro and, consequently, might be used in cell transplantation strategies for genitourinary reconstruction.
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Urothelial Cell
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We selected a 4-stain immunopanel including thyroid transcription factor (TTF)-1, cytokeratin (CK) 7, 34βE12, and CD56/neural cell adhesion molecule (NCAM) to subclassify a series of 45 pulmonary large cell carcinomas (LCCs) on bronchial biopsy. All cases consisted of a large tumor cell proliferation with abundant cytoplasm, vesicular nuclei, and prominent nucleoli. Immunohistochemically, 27 tumors (60%) were subclassified as adenocarcinoma (TTF-1+/CK7+, 24; CK7+ only, 3), 10 (22%) as squamous cell carcinoma (34βE12+ only), and 4 (9%) as LCC with neuroendocrine differentiation (CD56+, variably stained with TTF-1 and CK7, 34βE12–). In 4 cases, the tumors coexpressed CK7 and 34βE12 (3 cases) or were completely unstained (1 case). Surgically resected tumors matched exactly with the corresponding original biopsy specimens in 21 of 23 cases; consistent CD56 expression was a reliable marker in confirming a diagnosis of large cell neuroendocrine carcinoma even on biopsy. Our results suggest that the proposed 4-stain set of commercially available markers might help subclassify LCC even in small biopsy material, validating expression-profiling studies aimed at lung cancer classification and permitting more consistent patient enrollment for trials with targeted treatments.
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Cancer risk prognosis could improve patient survival through early personalized treatment decisions. This is the first systematic analysis of the spatial and prognostic distribution of different pan cytokeratin immunostaining intensities in breast tumors. The prognostic model included 102 breast carcinoma patients, with distant metastasis occurrence as the endpoint. We segmented the full intensity range (0–255) of pan cytokeratin digitized immunostaining into seven discrete narrow grey level ranges: 0–130, 130–160, 160–180, 180–200, 200–220, 220–240, and 240–255. These images were subsequently examined by 33 major (GLCM), fractal and first-order statistics computational analysis features. Interestingly, while moderate intensities were strongly associated with metastasis outcome, high intensities of pan cytokeratin immunostaining provided no prognostic value even after an exhaustive computational analysis. The intense pan cytokeratin immunostaining was also relatively rare, suggesting the low differentiation state of epithelial cells. The observed variability in immunostaining intensities highlighted the intratumoral heterogeneity of the malignant cells and its association with a poor disease outcome. The prognostic importance of the moderate intensity range established by complex computational morphology analyses was supported by simple measurements of its immunostaining area which was associated with favorable disease outcome. This study reveals intratumoral heterogeneity of the pan cytokeratin immunostaining together with the prognostic evaluation and spatial distribution of its discrete intensities.
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In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain. Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3'-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al.. (2012).
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Immunohistochemical staining was performed on 145 biopsies with a diagnosis of undifferentiated or poorly differentiated tumor in order to classify them into lymphoid, epithelial, or mesenchymal in origin. It was possible to arrive at a histogenetic diagnosis on immunostaining in 85.5% of cases. Immunostaining confirmed the diagnosis in 32.4% and contributed to diagnosis in 53.1%. Malignant lymphoma was the most common diagnosis (35.9%), followed by carcinoma (23.4%). A panel of antibodies consisting of anti-common leucocyte antigen (LCA), anti-epithelial membrane antigen (EMA), anti-cytokeratin (CK), anti-low to intermediate molecular weight cytokeratin (CAM 5.2), anti-S-100 protein (S-100), and anti-vimentin (VM) may resolve, to a large extent, some of the common diagnostic problems.
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Aberrant expression of CK20/CK7 is reported in a percentage of colorectal carcinomas (CRC); however, its relation to clinicopathological variables and survival data is still unclear. The objective of this study is to explore patterns of CK20/CK7 immunostaining in CRC and to analyse the diagnostic, prognostic, and predictive role of patterns of CK20/CK7 immunostaining.A total of 144 CRC cases were retrieved from the archives at the Department of Pathology, King Abdulaziz University, Jeddah, Saudi Arabia. Immunohistochemistry was performed using antibody to CK7 and CK20. Immunostaining was defined as low and high by using the extent of staining. The association of CK7 and CK20 with clinicopathological characteristics and survival.CK20 was expressed in a higher percentage of CRC and nodal metastasis than CK7. No difference in CK7 and CK20 immunostaining in primary and metastasis carcinomas was found. Four patterns of CK20/CK7 were identified; CK20+/CK7- (60.4%), CK20+/CK7+ (2.1%), CK20-/CK7- (35.4%), and CK20-/CK7+ (2.1%). There was no statistically significant correlation between CK20/CK7 immunohistochemical profile and clinicopathological characteristics, prognosis, and survival was determined.Our results may support the heterogeneity of CRC. CRC showed four different subclasses following patterns of relative CK20/CK7 immunostaining. A considerable number of CRC expressed aberrant immune profile of CK20/CK7, which should be considered during diagnosing CRC in metastatic regions. Further studies on larger cohorts correlating different immunohistochemical cytokeratin profiles to molecular subtypes of CRC are recommended for better understanding of pathogenesis and behaviour of CRC.
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Keratin 7
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Adenocarcinomas of uncertain origin are a frequent problem for surgical pathologists. To determine the utility of immunostaining for cytokeratin 7 and cytokeratin 20 in the separation of pulmonary adenocarcinomas from colonic adenocarcinomas, we studied routinely processed, formalin-fixed tissue from 151 of these tumors using commercially available monoclonal antibodies and an avidin-biotin immunohistochemical technique. Used alone, neither cytokeratin 7 immunostaining or cytokeratin 20 immunostaining reliably separated these tumors. However, the immunophenotype of cytokeratin 7 positive/cytokeratin 20 negative was seen in 86% of the pulmonary adenocarcinomas, and in 0% of the colonic adenocarcinomas. Conversely, the cytokeratin 7-negative/cytokeratin 20-positive immunophenotype was seen in 77% of the colonic carcinomas, and in 0% of the pulmonary tumors. In conclusion, cytokeratin 7/cytokeratin 20 immunostaining patterns may be helpful in separating pulmonary adenocarcinomas from colonic adenocarcinomas.
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Immunophenotyping
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