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Hyperdiploid pattern showing whole chromosomal gain, typically seen in biologically favorable neuroblastoma (Schwannian stroma-poor) tumor.

Diploid pattern often seen in biologically unfavorable tumor. The tumor of this case also has amplified MYCN.

Fluorescence in situ hybridization A neuroblastic rosette with nuclei containing an equal number of MYCN signals (in green) and signals of the reference probe (in red; located on chromosome 2). This pattern indicates the presence of predominantly three chromosomes 2 and a normal MYCN status

Fluorescence in situ hybridization for analysis of the MYCN status Neuroblastoma cells with a low to high amplification rate of the MYCN gene (over 4- up to 50-fold in relation to the reference probe). Some nuclei have a so-called MYCN gain which is defined as an up to 4-fold excess of gene copies as compared with the reference probe on chromosome 2. The gain or amplification of the MYCN oncogene is organized extrachromosomally in so-called double minute, i.e. acentric, chromosomes.

Fluorescence in situ hybridization for analysis of the MYCN status A neuroblastoma cell with MYCN amplification organized intrachromosomally (known as homogenously staining regions, HSR‘s) surrounded by bone marrow cells.

Fluorescence in situ hybridization for analysis of the MYCN status Neuroblastoma cells with MYCN amplification. The amplified oncogene is present in forms of double minutes and small HSR‘s.

Fluorescence in situ hybridization for analysis of the MYCN status The four pictures are derived from the same tumor and show an intra-tumoral heterogeneity for the MYCN status, i.e. a focal MYCN amplification. Besides nuclei with three single copy MYCN signals, there are nuclei with up to 60 signals.

Fluorescence in situ hybridization for analysis of the MYCN status A neuroblastoma metastasis in the liver showing MYCN amplified cells besides nuclei with a so-called MYCN gain (for definition see Figure 1)

Immunofluorescence labeling and sequential fluorescence in situ hybridization with analysis of the MYCN status A. Neuroblastoma cells in the bone marrow stained with an anti-GD2 antibody. B. The sequential FISH with a MYCN probe and automatic relocation of the GD2 positive cells (shown in A) revealed a MYCN amplification (HSR‘s).

Fluorescence in situ hybridization for analysis of the chromosomal region 1p36.3 A rosette consisting of poorly differentiated neuroblasts showing three green signals for the centromeric region of chromosome 1 and three red signals for the subtelomeric region of the short arm of chromosome 1 (1p36.3). The balanced signal ratio indicates an intact chromosomal region 1p36.3. One cell shows a hexasomy 1.

A ganglionic cell with one big centromeric signal and three subtelomeric signals. The big signal is caused by centromeric associations which are commonly observed when neuroblastoma cells differentiate into ganglionic cells, but can also be found after cytotoxic therapy. Centromeric associations, especially with polyploidization, may hamper correct interpretation of the FISH picture (a deletion or imbalance of a chromosomal region can be masked, or a gain or, rarely, an amplification can be feigned).

The same probes as shown in A. and B. indicate a so-called ‚imbalance‘ of the chromosomal region 1p36.3 (mainly three to six centromeric signals and 2 to three subtelomeric signals). Although in neuroblastic tumors this hybridization pattern is often a consequence of a loss of heterozygosity (LOH), it does not necessarily reflect an LOH. Some nuclei show fusion of centromeric signals which masks the underlying imbalance .

A hybridization pattern showing only one 1p36.3 signal is called ‚deletion‘ and is always connected with an LOH.

Fluorescence in situ hybridization of ganglionic cells and Schwann cells in ganglioneuroblastoma, intermixed tumor Ganglionic cells with trisomies for the chromosome 1 without deletion or imbalance of the chromosomal region 1p36.3. Trisomies to hexasomies of various chromosomes are usually found in the neuronal population of spontaneously maturing neuroblastic tumors. The DNA content is usually in the near-triploid (near-penta-, hexaploid) range.

Schwann cell nuclei showing two centromeric and two 1p36.3 signals indicating the presence of a disomy 1. The same hybridization pattern is found with all other chromosomes which are investigated. Structural aberrations are always absent and the DNA content of the Schwann cell population in these tumor is constantly diploid. The genetic differences of these two cell populations, i.e., neuroblastic/ganglionic cells and Schwann cells, the results of co-cultivation studies, and embryological/neurophysiological data lead to the conclusion that Schwann cells in pNTs are normal and are recruited by the neuroblastoma cells.

Fluorescence in situ hybridization for the analysis of the status of the long arm of chromosome 17 The chromosome 17q status is analyzed with a DNA probe located on the long arm of chromosome 17 (in green) together with a reference DNA probe located on the short arm of chromosome 17 (in red). This picture shows a balanced ratio of 17p and 17q signals and indicates a tetrasomy 17.

Fluorescence in situ hybridization for the analysis of the status of the long arm of chromosome 17 The chromosome 17q status is analyzed with a DNA probe located on the long arm of chromosome 17 (in green) together with a reference DNA probe located on the short arm of chromosome 17 (in red). This picture shows a balanced ratio of 17p and 17q signals and indicates Different types of chromosome 17q gains are shown.

Genetics

In this section, genetic properties of pNTs are illustrated by conventional karyotyping and FISH method


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