Aspiration cytology of neuroblastoma: Light microscopy with transmission and scanning electron microscopic correlations
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Abstract Fine‐needle aspiration biopsies from three patients with neuroblastoma were studied by light microscopy, and the morphologic findings were correlated with those from transmission and scanning electron microscopy. Light microscopic examination of the aspiration smears from all three cases revealed small and large round cells with variable numbers of intertwining cytoplasmic processes. Transmission electron microscopy confirmed the light microscopic finding of cytoplasmic processes; in addition, it revealed the presence of other diagnostic morphologic features, including neurosecretory granules, microtubules, and synaptic cell junctions. Scanning electron microscopy demonstrated that the tumors were composed of a mixture of undifferentiated round cells and more differentiated cells with long cytoplasmic processes. The morphologic spectrum of these processes and their interrelationships with one another and with other cells could be studied in detail. These findings indicate that scanning electron microscopy may be used effectively in the morphologic evaluation and pathologic diagnosis of neuroblastoma.A septum-rich fraction was prepared from Triehophyton mentagrophytes, and the ultrastructural investigation of septa was carried out by shadowing and thin section electron microscopy before and after enzymatic and/or alkaline treatments. Tri-lamellar structure, consisting of an electron lucid middle layer and two outer electron dense layers, was observed by thin section electron microscopy. By shadowing electron microscopy, the surface ultrastructure of septa exhibited a randomly orientated microfibrillar network structure, which may correspond to the electron dense outer layers of the septum observed in thin section preparations. However, shadowed septa after papain digestion revealed a spiral arrangement of microfibrils consisting of rodlet-like units, which disappeared during chitinase treatment. This spiral structure may therefore be composed primarily of chitin. It was suggested that this spirally arranged microfibrils may correspond to the electron lucid middle layer observed in thin section preparations.
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Morphology
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Neuroblastoma is a pediatric malignant tumor of the postganglionic sympathetic nervous system that usually develops in the adrenal gland or in nonadrenal abdominal or thoracic sites.[1] It is the most common malignancy in infants and the most common extracranial solid tumor of childhood, with approximately 650 cases diagnosed annually in the United States.[2] The dramatic age-related survival differences among neuroblastoma patients with a similar tumor stage emphasize the heterogeneity of neuroblastoma pathobiology. Early research efforts to understand the pathobiology of neuroblastoma[3-5] and the significant progress made in neuroblastoma molecular biology[6] have informed the clinical treatment of neuroblastoma.
Solid tumor
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A comparative ultrastructural study was carried out on several species of Actinomyces and related Gram‐positive rods including Arachnia, Bacterionema, Rothia and Leptotrichia . A total of 52 well characterized strains were examined by transmission electron microscopy. Particular attention was paid to the ultrastructure of the cell periphery, that is the plasma membrane, periplasmic space, the cell wall per se and extramural structures, including surface “fuzz”. In addition, the ultrastructure of various features of the cytoplasm were also examined. A good correlation appeared to exist between certain ultrastructural characteristics of the microorganisms and their taxonomic position as determined by other criteria. It should be noted, however, that the ultrastructure of certain strains differed markedly from that of the remaining strains of the species. This observation raises some doubt on the appropriateness of the current classification for these particular strains. The ultrastructural features of the cell periphery were found to be particularly stable for all strains grown under standard conditions. For this reason, ultrastructural features of the cell wall and associated structures are probably a more reliable source of morphological criteria for identification purposes than the ultrastructural characteristics of the cytoplasmic components which tend to be more variable. The results suggest that certain ultrastructural features are useful criteria for the identification and classification of these Gram‐positive rods.
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Abstract MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified. (Cancer Res 2006; 66(5): 2826-33)
N-Myc
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Abstract Light has its limits, and even in the world of ‘super resolution’ microscopy, many cellular structures and even large protein complexes are not resolvable in the light microscope – although it is ideal for watching living cells and tissues and linking structural changes to functional outcomes. Electron microscopy can yield molecular level resolution but is limited to nonliving samples that have been extensively processed to preserve structure and be visible using electron optics. While it would be ideal to have a single system which provided nanoscale information on living samples, until that day arrives, the obvious answer is to use both light microscopy and electron microscopy as complementary approaches. Localising the same labelled structure in both imaging modalities is known as correlative LM and EM or correlated light and electron microscopy. Various methods and instrumentation are used for correlating data from the same sample imaged using both light and electrons. Key Concepts Light microscopes cannot resolve specimens at the nanoscale. Combining light and electron microscopy can link ultrastructure to function. For CLEM, exactly the same region of interest (ROI) is imaged in both the light and electron microscope. 3D (volume) electron microscopy can provide spatial information at the nanoscale. Using special probes, proteins can be located in both light and electron microscopes.
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The prognosis of mediastinal neuroblastoma has been reported to be better than for other neuroblastomas. The reason for this is however not clear. Furthermore, a comparison between mediastinal neuroblastoma and the other neuroblastomas has been rarely reported so far. In this study, the characteristics of mediastinal neuroblastoma (84 cases) are investigated and compared with those of other neuroblastomas (440 cases). Regarding clinical factors, the age distribution and the rate of cases detected at mass screening were similar in both groups. According to Evan's staging system, the rates of early stage (I, II) were 62% in the mediastinal neuroblastoma and 38% in the other neuroblastomas (p<0.001). Regarding the biological prognostic factors, a favorable histology based on Shimada's classification was found in 100% (35/35) of the mediastinal neuroblastoma cases and in 85% (112/132) of the other neuroblastoma cases (p<0.05). With regard to N-myc amplification, all of the examined 42 cases in mediastinal neuroblastoma had a N-copy number of less than 10 copies, while 32 of the examined 263 cases (12%) in the other neuroblastomas had an amplification of N-myc of more than 10 copies (p<0.05). The 5-year survival rates were 78% in the mediastinal neuroblastoma and 59% in the other neuroblastomas, respectively. Of the cases who underwent an incomplete resection of primary tumors in localized neuroblastoma, the 5-year survival rate of the mediastinal neuroblastoma cases was significantly more favorable than that of the other neuroblastomas. The majority of mediastinal neuroblastoma cases showed an early stage and favorable prognostic factors. It is likely that the clinical and biological prognostic factors of the tumor are therefore more closely correlated with the outcome of mediastinal neuroblastoma rather than the degree of the surgical resection. Regarding the treatment for mediastinal neuroblastoma, it is most important to evaluate the biology of the tumor after surgical resection.
Histology
Mediastinal tumor
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1. A genomic amplification of N-myc of neuroblastoma was frequently observed in patients in the advanced stage of the disease, in those with the tumor originating from the suprarenal region, and in those with a histologically undifferentiated neuroblastoma. Thus, N-myc may be one of the most pertinent prognostic factors of neuroblastoma in patients over one year of age. 2. The neuroblastoma patient with 1-10 copies of N-myc responded to aggressive multidisciplinary therapy, even those over one year of age. 3. Rapid invasion and progression of the tumor was evident in children with more than 10 copies of N-myc. 4. N-myc amplification may correlate with immaturity of catecholamine metabolism of neuroblastoma.
N-Myc
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Immunostaining
Immunoelectron microscopy
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The high-resolution observation of specimens in their natural state has long been a goal of microscopy. In electron microscopy, the dogma has been that extensive stabilization of the specimen is required to withstand the specimen-beam interactions and high vacuum conditions inside the microscope column. Cryo-electron microscopy, the observation of specimens at low temperatures, is a relatively young branch of microscopy. The preparation of a sample for cryo-electron microscopy involves only a few steps as outlined below. Compared to "standard" procedures for electron microscopy that mostly involve chemical fixation and the dehydration with organic solvents and often more or other chemical treatments, cryo-electron microscopy offers a tremendous reduction in the number of preparatory steps. A thin film may form spontaneously between the bars of a specimen grid for electron microscopy when such a grid is dipped and withdrawn from a suspension.
Cryo-Electron Microscopy
Cryofixation
Polymer characterization
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