Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma

Strehl, Sabine; Ambros, Peter F.

doi:10.1159/000133494

Cited by 60 publications

(33 citation statements)

References 3 publications

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“…To enable the visualization of these aberrations in interphase nuclei and to confirm classical cytogenetic analysis, doubletarget FISH using heterochromatin-specific probes for chromosomes 1 and 16 and the sub-telomeric midi-satellite probe specific for locus D1Z2 is a rapid and reliable method [36,41]. Deletions at 1p36.3 are indicated by two D1Z1 hybridization signals and one D1Z2 signal in diploid cells ( fig.…”

Section: ➝ ➝mentioning

confidence: 99%

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information

2000

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Ewing tumors (ETs) are characterized by rearrangements of the EWS gene located in 22q12 and a high MIC2 expression. In 85% of ETs, the t(11;22)(q24;q12) generates chimeric fusion transcripts between the EWS and the FLI1 gene, whereas in the remaining cases the EWS gene is rearranged with different partners of the ETS oncogene family. Besides classical cytogenetic analysis, fluorescence in situ hybridization (FISH) and RT-PCR can be used to demonstrate these 22q12 rearrangements which are pathognomonic for ETs. To visualize 22q12 rearrangements in individual cells, DNA probes flanking the EWS-R1 breakpoint region on chromosome 22 can be hybridized in double-target FISH experiments on tumor cell preparations. Intact chromosomes 22 are indicated by juxtaposition of the DNA probes, whereas rearrangements of the EWS gene separate the hybridization signals. In addition to 22q12 rearrangements, numerical aberrations of chromosomes 8 and 12 can be observed in about 50% of ETs, deletions at the short arm of chromosome 1 and der (16)t(1;16)(q12;q11.2) chromosomes in about 20% of the cases. Numerical aberrations, deletions at 1p36.3, and the t(1;16) were detected by using double-target FISH on touch, cytospin, and chromosome preparations, on frozen and paraffin sections and isolated deparaffinized nuclei. So far, numerical aberrations of chromosomes 8 and 12 did not show prognostic impact. However, deletions at 1p36.3 and imbalances between the long and short arms of chromosome 1 were associated with adverse clinical outcome in a group of patients with localized disease.

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Section: ➝ ➝mentioning

confidence: 99%

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information

2000

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“…FISH allows rapid and accurate determination of MYCN copy number, allows MYCN copy number evaluation at the single cell level (5) and can be performed on tumor imprints of biopsies that can be evaluated for tumor cell morphology and content by combination with immunohistochemical staining (6). Despite the high sensitivity and specificity of FISH for detection of MNA, errors in assessment of MYCN copy number, albeit rare, may occur.…”

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confidence: 99%

Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay

et al. 2002

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Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for timeconsuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C T method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis.

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“…From this, we conclude that the APase-Fast Red reaction product does not make the cells less accessible to ISH reagents and does not shield the target DNA. The latter phenomenon has been shown to occur when the peroxidase-diaminobenzidine reaction is used for the ICC step (18,23). Application of the APase-Fast Red reaction has the advantage that directly FITClabeled, centromere-specific DNA probes can thus be detected without any amplification step ( Figures IG, 1H, 11, and lK), which increases analytical speed and reduces background reactions.…”

Section: Discussionmentioning

confidence: 99%

Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells.

Speel¹,

Herbergs²,

Ramaekers³

et al. 1994

J Histochem Cytochem.

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We describe the development and application of a sensitive high-resolution fluorescence alkaline phosphatase (&e)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluoresm c e intensity, accurate localization, and advantageous slowfading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, &e-FastRed immunostaining could be combined with subsequent visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H8Z and EPLC 65 were used as a

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Fluorescence in situ hybridization combined with immunohistochemistry for highly sensitive detection of chromosome 1 aberrations in neuroblastoma

Cited by 60 publications

References 3 publications

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information

Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay

Combined immunocytochemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells.

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