Abstract
Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.
Highlights
A variety of methods are available for the detection of genetic aberrations in the routine diagnostic work-up of tumor specimens
It was of considerable interest to us to perform a comprehensive study in order to meticulously assess the reliability of fluorescence in-situ hybridization (FISH) analysis in a high-throughput tissue microarrays (TMAs) setting for the widely used break-apart probes, but especially for the discovery of deletions in formalin-fixed paraffin-embedded (FFPE) tissue samples
We analyzed 46 B-NHL samples with known karyotypes spotted on TMAs for hallmark lymphoma-associated translocations of the IGH, BCL2, BCL6and MYC-genes
Summary
A variety of methods are available for the detection of genetic aberrations in the routine diagnostic work-up of tumor specimens. Classical chromosome banding and fluorescence in-situ hybridization (FISH) are of major importance. Conventional cytogenetic approaches are still considered to be the gold standard in genetic diagnostics, allowing for the comprehensive analysis of tumor-specific alterations in the karyotype, banding analyses are limited by the availability of fresh material and by the need for in-vitro cultivation of tumor cells. A major advantage of FISH assays is the possibility to perform hybridizations in high-throughput approaches using tissue microarrays (TMA) in diagnostic settings, and especially for retrospective studies of archival pathological specimens. A number of these high-throughput studies have been performed on tumor specimens and it is generally agreed that translocations and gene amplifications can be readily detected in FFPE TMA formats [3,4,5]. A meticulous analysis of the reliability of the results of translocation analyses on TMAs, has not been convincingly done so far, and it is still a matter of debate whether deletions can be reliably detected using the TMA approach
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