New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

Anti-cancer effects of dioscin on three kinds of human lung cancer cell lines through inducing DNA damage and activating mitochondrial signal pathway.

Dioscin, a natural steroid saponin, has been widely investigated. However, its anti-cancer activities on human lung cancer cells are still unknown. In the present paper, the inhibitory effects of dioscin were investigated, and the results showed that dioscin inhibited the proliferation of human A549, NCI-H446 and NCI-H460 cancer cells. DNA damage and cell apoptosis in dioscin-treated cells were found through single cell gel electrophoresis and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling assays. Furthermore, dioscin caused mitochondrial structure changes and blocked cell cycle at S phase based on transmission electron microscope and flow cytometry analysis. In addition, dioscin treatment caused the release of cytochrome c from mitochondria into cytosol. The activities of Caspase-3 and -9 in dioscin-treated groups were significantly increased compared with control group. Western blotting analysis showed that dioscin significantly down-regulated the expressions of Bcl-2 and Bcl-xl, and up-regulated the expressions of Bax, Bak and Bid. Our results indicate that dioscin has anticancer activities against human lung cancer cells through inducing cell cycle arrest, DNA damage and activating mitochondrial signal pathway.

Using electron microscopy and multivariate cluster analysis to determine diagnosis and prognosis in cases of neuroendocrine lung carcinoma.

OBJECTIVE: To establish reproducible electron microscopic criteria for identifying the four major types of neuroendocrine tumors of the lung: carcinoid; atypical carcinoid; large cell neuroendocrine carcinoma; and small cell carcinoma. METHODS: Measurements were made on electron micrographs using a digital image analyzer. Sixteen morphometric variables related to tumor cell differentiation were assessed in 27 tumors. The examination under electron microscopy revealed that all of the tumors could be classified as belonging to one of the four categories listed above. Cluster analysis of the morphometry variables was used to group the tumors into three clusters, and Kaplan-Meier survival function curves were employed in order to draw correlations between each cluster and survival. RESULTS: All three clusters of neuroendocrine carcinomas were found to be associated with survival curves, demonstrating the prognostic significance of electron microscopic features. The tumors fell into three well-defined clusters, which represent the spectrum of neuroendocrine differentiation: typical carcinoid (cluster 1); atypical carcinoid and large cell neuroendocrine carcinoma (cluster 2); and small cell carcinoma (cluster 3). Cluster 2 represents an intermediate step in neuroendocrine carcinogenesis, between typical carcinoid tumors and small cell carcinomas. CONCLUSIONS: Our findings confirm that electron microscopy is useful in making the diagnosis and prognosis in cases of lung tumor.

Large cell variant ovarian small cell carcinoma: case report.

Ultrastructural data about large cell variant ovarian small cell carcinoma (LCV-SCC) are scarce and contradictory and the role of transmission electronmicroscopy (TEM) is not clear in the assessment of such tumors. The authors present a case of LCV-SCC without hypercalcemia in a 30-year-old woman. The diagnosis was confirmed by histopathological and immunohistochemical studies. Cytopathological examination of peritoneal washing showed a population of large neoplastic cells. TEM demonstrated that the neoplasia comprised two types of cells: one type showed many coarse secretory granules without dense core, and the other type was without granules and showed dilated endoplasmic reticulum and sometimes indented nuclei. The present case indicates that different underlying ultrastructural patterns, not yet well known, exist in connection with the pathological and clinical behaviour of LCV-SCC. TEM might play a role in the identification of subtypes of LCV-SCC with different prognostic and therapeutic impact.

Diagnóstico e prognóstico dos tumores pulmonares neuroendócrinos mediante microscopia eletrônica e análise multivariavel de agrupamento / Using electron microscopy and multivariate cluster analysis to determine diagnosis and prognosis in cases of neuroendocrine lung carcinoma

OBJECTIVE: To establish reproducible electron microscopic criteria for identifying the four major types of neuroendocrine tumors of the lung: carcinoid; atypical carcinoid; large cell neuroendocrine carcinoma; and small cell carcinoma. METHODS: Measurements were made on electron micrographs using a digital image analyzer. Sixteen morphometric variables related to tumor cell differentiation were assessed in 27 tumors. The examination under electron microscopy revealed that all of the tumors could be classified as belonging to one of the four categories listed above. Cluster analysis of the morphometry variables was used to group the tumors into three clusters, and Kaplan-Meier survival function curves were employed in order to draw correlations between each cluster and survival. RESULTS: All three clusters of neuroendocrine carcinomas were found to be associated with survival curves, demonstrating the prognostic significance of electron microscopic features. The tumors fell into three well-defined clusters, which represent the spectrum of neuroendocrine differentiation: typical carcinoid (cluster 1); atypical carcinoid and large cell neuroendocrine carcinoma (cluster 2); and small cell carcinoma (cluster 3). Cluster 2 represents an intermediate step in neuroendocrine carcinogenesis, between typical carcinoid tumors and small cell carcinomas. CONCLUSIONS: Our findings confirm that electron microscopy is useful in making the diagnosis and prognosis in cases of lung tumor.

OBJETIVO: Estabelecer, com ajuda do microscópio eletrônico, critérios que possibilitem uma diferenciação mais exata entre os quatro tipos maiores de tumores neuroendócrinos pulmonares: tumor carcinóide típico e atípico, carcinoma de grandes células neuroendócrino e carcinoma de pequenas células. MÉTODOS: Todos os tumores foram avaliados morfometricamente e 16 variáveis foram relacionadas com diferenciação das células tumorais; estas variáveis foram analisadas sob microscopia eletrônica com ajuda de um analisador de imagem digital em 27 tumores. A avaliação através da microscopia eletrônica revelou que todos os tumors investigados podiam ser classificados a um dos quarto tipos listados acima. A análise das variáveis morfométricas foi usada para agrupar os tumores em três grandes grupos, os quais foram relacionados à sobrevivência pelas curvas de Kaplan Meier. RESULTADOS: Os três grupos de carcinoma neuroendócrino associaram-se às curvas da sobrevivência, as quais mostraram características ultrastruturais na microscopia eletrônica de significância prognóstica distinta. Os tumores foram contidos em três grupos bem definidos, que representam o espectro da diferenciação neuroendócrina: tumor carcinóide (grupo 1); tumor carcinóide atípico e carcinoma de grandes células neuroendócrino (grupo 2); e carcinoma de pequenas células (grupo 3). O grupo 2 representa um espectro intermediário na carcinogênese neuroendócrina, entre o carcinóide típico e o carcinoma de pequenas células. CONCLUSÕES: Nossos achados confirmam que a microscopia eletrônica é uma ferramenta útil no diagnóstico e prognóstico dos casos de tumores pulmonares.

Expression of neuroendocrine markers in non-small cell lung cancer.

Neuroendocrine (NE) differentiation is reported in some cases of non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the expression of NE markers in NSCLC using novel sensitive methods. 20 cases of NSCLC were examined using immunohistochemical (IHC) and immunoelectron microscopy (IEM) methods. In addition, circulating levels of the NE markers chromogranin A (CgA) and neuron-specific enolase (NSE) were measured. Using conventional IHC methods, two tumours (10%) showed immunoreactivity for synaptophysin (SYN), one (5%) for Cg and four (20%) for neural cell adhesion molecule (NCAM). Adding the tyramide signal amplification (TSA) technique, the number of immunoreactive tumours for both SYN and CgA increased to five (25%). No increased immunoreactivity was achieved for NCAM. Nine tumours (45%) were immunoreactive for SYN, CgA or NCAM. Using IEM, one of five representative samples that revealed IHC reactivity for CgA showed immunogold labelling of CgA in cytoplasmic vesicles. Elevated levels of circulating CgA or NSE did not correlate with positive IHC findings. In conclusion, using sensitive IHC methods NE differentiation was seen in a greater proportion of NSCLC than previously reported. Sensitive methods may improve our understanding of the tumour biology and represent an important diagnostic tool for future therapeutic modalities.

Cytokeratin 20-positive large cell neuroendocrine carcinoma of the colon.

Herein is presented a case of cytokeratin (CK) 20-positive large cell neuroendocrine carcinoma of the colon, in which the tumor was clinically at stage IV and located in the ascending colon. Pathological examination of the resected tumor revealed nested and solid proliferation of large undifferentiated cells with vesicular nucleus and prominent nucleoli. No areas showed differentiation toward adenocarcinoma or squamous cell carcinoma. Tumor cells were immunohistochemically positive for chromogranin A, synaptophysin, CD 56 (focal), and bore electron-dense granules. With these features, the tumor was diagnosed as a large cell neuroendocrine carcinoma of the colon. Liver metastasis and local recurrence progressed, and the patient died of the primary disease 7 months after operation. The autopsy confirmed this diagnosis without detectable tumors in the lungs. Interestingly, more than half of the tumor cells were positive for CK 20, while CK 7 was not expressed. Most neuroendocrine carcinomas do not express CK 20, with the exception of Merkel cell carcinomas, and most colorectal adenocarcinomas express CK 20. To the best of the authors' knowledge, the present case is the first CK 20-positive, CK 7-negative colorectal neuroendocrine carcinoma to be described, suggesting a link between colorectal neuroendocrine carcinoma and conventional adenocarcinoma.

Combined large cell neuroendocrine carcinoma and spindle cell carcinoma of the lung.

We report a unique case of a combined pulmonary large-cell neuroendocrine carcinoma and spindle-cell carcinoma. The patient was a 54-year-old female smoker who presented with a 4-month history of increased left-sided chest pain and exertional dyspnea. The left upper lobectomy specimen revealed an 8.0-cm mass with central necrosis. Microscopically, the epithelial areas were composed of well-defined nests of large cells with peripheral palisading expressing low-molecular-weight keratin, synaptophysin, chromogranin, and neuron-specific enolase. The spindle-cell component consisted of pleomorphic cells arranged in fibrosarcoma and malignant fibrous histiocytoma-like patterns. These spindle cells were positive for low-molecular-weight keratin and vimentin with focal expression of CD68 and muscle-specific actin. Electron microscopy in the spindle-cell areas showed cell junctions and numerous tonofilaments, indicative of epithelial differentiation. The tumor behaved aggressively and the patient died with extensive metastases 4 months after surgery. The combination of neuroendocrine malignancies and spindle-cell carcinomas appears to be uncommon in the lung. Previous reports have described this association in single case reports of anaplastic small-cell carcinoma and atypical carcinoid, but not in large-cell neuroendocrine carcinoma.

Epithelial tumors of the lung.

Our knowledge and understanding of bronchopulmonary tract tumors have grown considerably; modern pathology enables the phenotyping of many tumors with increasingly improving techniques and tools and, arguably, improving criteria. By the same token, at least some of the new data may not be readily grafted onto traditional classification schemes. Some traditional designations will be dropped and replaced. And, although it has been overenthusiastically argued that molecular classifications may be attained, that ideal might not be truly an improvement. For classifications to be useful, they should be relatively simple, easily reproducible, and clinically significant. Still, modern marker pathology has revealed new vistas for the evaluation, diagnosis, and therapy of at least some tumors. These developments merit optimism but also caution from clinicians and pathologists.

Tomography of cells by confocal laser scanning microscopy and computer-assisted three-dimensional image reconstruction: localization of cathepsin B in tumor cells penetrating collagen gels in vitro.

We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membrane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function.

Activity and tissue localization of cathepsin G in non small cell lung cancer.

Activity and tissue localization of cathepsin G were examined in tumors deriving from 73 patients with non small cell lung cancer. Activity of cathepsin G was highest in adenocarcinoma, lower in planoepitheliale cancer, the lowest in macrocellular cancer. In all histological types of tumors cathepsin G activity in supernatants was lower than in sediments. The enzyme immunohistochemically was localized in neutrophils. There is evident correlation between neutrophil numbers and cathepsin G activity in examined cancer types. Result of our examinations indicate a relationship between cathepsin G activity, grade of tumor differentiation and particular clinical stages of disease.

Mixed small cell/large cell carcinoma of the lung. Report of a case with cytologic features and ultrastructural correlation.

BACKGROUND: Mixed small cell/large cell carcinoma of the lung, a chemotherapy- and radiation-resistant subtype of small cell lung cancer, constitutes 4-6% of small cell lung cancer but has been described rarely in the cytopathology literature. CASE: A 64-year-old man presented with a left hilar mass. A concurrent transbronchial biopsy and postbiopsy bronchial washing were performed. The latter presented as loosely cohesive cells, scattered singly, in small clusters, in monolayer sheets and in a perivascular arrangement. The tumor exhibited a wide spectrum of cytomorphology: small cells with pyknotic nuclei and scanty cytoplasm were admixed with larger cells with vesicular nuclei, prominent nucleoli and abundant cytoplasm. Nuclear shape included oval, spindle and peg. Nuclear size was also highly variable. The tumor was retrieved from the transbronchial biopsy and processed for ultrastructural study. CONCLUSION: Based on cytologic-ultrastructural correlations. the seemingly "mixed" morphology may have resulted from the rapid degeneration of a single clone of tumor cells: the viable tumor cells were the "large" cells, and the dying cells were the "small" cells. A novel ultrastructural observation is that the perivascular tumor cells developed peculiar, fingerlike cytoplasmic processes abutting an undulating basement membrane along the thin-walled blood vessel.

[Large cell carcinoma of the lung: ultrastructural classification, diagnosis, prognosis]. / Krupnokletochnyi rak legkogo: ul'trastrukturnaia klassifikatsiia, diagnostika, prognoz.

16 lung large cell carcinomas (13 of them were followed for a long time) were studied. It is established that these tumours are a heterogeneous group of neoplasms which may be undifferentiated or with squamous, glandular, oncocytic and endocrine differentiation. Ultrastructural classification is presented reflecting diagnostic and prognostic aspects of these tumours. Tumours with pneumocytes type II, oncocytic differentiation and many lipid inclusions have more favourable prognosis. Unfavourable prognosis is typical for large cell carcinoma formed of undifferentiated cells only or with differentiation in the direction of goblet cells.