BACKGROUND:In malignant pleural mesothelioma (MPM), most patients first present with pleural effusion; thus, cytologic analysis is the primary diagnostic approach. However, the cytologic distinction between MPM and reactive mesothelial cells (RMCs) in effusions can be extremely difficult due to the lack of both well-established immunocytochemical markers and definite cytological criteria for MPM. Moreover, the existence of both MPM cells and RMCs in effusions from the same patient makes the differentiation even more challenging. Homozygous deletion of the 9p21 locus, the site of the cyclin-dependent kinase inhibitor 2A/p16 (CDKN2A/p16) gene, frequently occurs in MPM but has never been reported in RMCs. The aim of this study was to define the cytomorphological characteristics of MPM cells, identified by the presence of 9p21 homozygous deletion by fluorescence in situ hybridization (FISH). METHODS: For this purpose, cells on smear preparations were recorded using a virtual microscope system and were subjected to FISH analysis. Thereafter, 9p21 homozygous deletion-positive cells were identified in the recorded virtual slides, followed by analysis of their morphological characteristics. RESULTS: Mesothelioma cells positive for the 9p21 homozygous deletion exhibited significantly more frequent cellin-cell engulfment, multinucleation (more than 2 nuclei), and larger multicellular clusters composed of more than 10 cells than did 9p21 deletion-negative RMCs. Possible cutoff values are also proposed for these morphological markers to differentiate MPM cells from RMCs. CONCLUSIONS: These morphological differences and cutoff values are useful for cytological differentiation of mesothelioma cells from RMCs. In addition, the novel technique of a combination of virtual microscopy and FISH is introduced for tumor morphological analysis. Cancer (Cancer Cytopathol) 2013;121:415-22. V C 2013 American Cancer Society.KEY WORDS: pleural mesothelioma; cytology; fluorescence in situ hybridization; 9p21 homozygous deletion; p16.

There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.

Recently, a proteasome β subunit expressed exclusively in thymic cortical epithelial cells was discovered in mice and humans. This subunit, designated β5t, is a component of the thymoproteasome, a specialized type of proteasome implicated in thymic positive selection. To investigate whether β5t could serve as a marker for the differential diagnosis of thymic epithelial tumors, we performed immunohistochemical analysis using anti-β5t antibody in 54 cases of thymic epithelial tumors comprising 41 cases of thymomas and 13 cases of thymic carcinomas. β5t was detected in the neoplastic epithelial cells of thymomas. Among the subtypes of thymoma, expression of β5t was observed in most cases of type B thymoma (20 of 21) but not in type A thymomas (0 of 3). In type AB thymomas, β5t expression was variable (6 of 17). Type B3 thymomas (4 cases) were positive for β5t but negative for CD5, c-kit, and glucose transporter 1 (GLUT-1), which are known as diagnostic markers for thymic carcinomas. In contrast, thymic carcinomas were negative for β5t (0 of 13) but expressed at least one and usually all of CD5, c-kit, and GLUT-1. Thus, β5t and CD5/c-kit/GLUT-1 were differentially expressed in type B3 thymoma and thymic carcinoma. We tested β5t expression in 39 cases of tumors arising from other organs, which showed the specific expression of β5t in thymic epithelial tumors. This study demonstrates that β5t is expressed in most type B and in some type AB thymomas and is a marker useful in differentiating type B3 thymomas from thymic carcinomas when used in combination with other diagnostic markers.

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budding grade is associated with invasive clinicopathological features, adverse postoperative prognosis and EMT status in extrahepatic cholangiocarcinoma.