Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization

Our plain-English summary of Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization, by H. Horn, J. Bausinger, A.M. Staiger, et al (PloS One, 2014).

The study

• Authors: H. Horn, J. Bausinger, A.M. Staiger, M. Sohn, C. Schmelter, K. Gruber, C. Kalla, M.M. Ott, A. Rosenwald, G. Ott
• Journal: PloS One
• Published: 2014;9(4):e95047
• Cited by: 16 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory study tested how reliably fluorescence in-situ hybridization (FISH), a technique that marks specific genes so they can be seen under a microscope, detects genetic changes in archived tumor tissue. The tissue was arranged on tissue microarrays, which are small cores from many samples placed on one slide. The authors used 46 B-cell lymphoma samples with known chromosome results, 62 follicular lymphoma samples, six malignant mesothelioma samples, and mixtures of two lymphoma cell lines. Of 184 tests for gene rearrangements, 171 (93%) were readable, and 159 of those 171 (93%) agreed with chromosome banding. In the cell-line mixtures, 17% to 25% of cells showed false deletion-like signals caused by slicing through cell nuclei. In all six mesothelioma samples, FISH found CDKN2A deletions: one copy lost in two samples and both copies lost in four. A PCR test confirmed the four double losses when measured against one reference gene, but did not detect the single-copy losses. The authors concluded that FISH was reliable and more sensitive than PCR, provided strict cut-off values and appropriate controls are used. The study did not examine asbestos exposure.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory method-validation study comparing FISH on tissue microarrays with chromosome banding, FISH on whole tissue sections, and quantitative PCR, plus a cell-line dilution experiment
• Who was studied: Archived formalin-fixed paraffin-embedded tumor samples from Würzburg and Stuttgart, Germany: 46 B-cell lymphomas with chromosome banding data (24 primary nodal DLBCL, 11 extranodal DLBCL, 11 follicular lymphomas; banding done 1993 to 2007), 62 follicular lymphomas (10 with cytogenetic data), six malignant mesothelioma specimens (without cytogenetic data, site not stated), and the mantle cell lymphoma cell lines REC1 and JVM2
• Main results:
  • For translocation testing on tissue microarrays, 171 of 184 hybridizations (93%) were of adequate quality
  • FISH and chromosome banding agreed in 159 of 171 calls (93%); 9 of 171 changes were found only by FISH, and 3 of 171 (deletions and inversions of BCL6) only by chromosome banding
  • In the 25 samples re-tested on whole tissue sections, all 88 evaluable hybridizations matched the tissue microarray results
  • In cell-line mixtures, sectioning artefacts (cut nuclei giving false deletion-like signals) occurred in 17% to 25% of cells
  • In the six malignant mesothelioma samples, FISH showed monoallelic (one-copy) CDKN2A deletions in two samples (in 36% to 47% of cells) and loss of both alleles in four samples (in 32% to 75% of cells)
  • In the mesothelioma samples, qPCR confirmed all four double deletions when HBB was the reference gene and one when MAL was the reference gene; qPCR did not detect the monoallelic CDKN2A deletions
  • In follicular lymphoma, of 26 samples with ATG5 (6q21) deletion shown by FISH, only 13 showed the deletion by qPCR
• The authors' conclusion: The authors concluded that FISH was an excellent tool for detecting structural and numerical genetic aberrations in FFPE tissue on tissue microarrays. They stated that rigorous cut-off values and appropriate controls are required, and that the sensitivity of FISH exceeded that of qPCR.
• Limitations the authors state:
  • Secondary genetic lesions might not be representative of the whole tumor population
  • For qPCR, the authors could not exclude copy number changes in the reference gene regions, which may have affected measurements
  • DNA from FFPE specimens is often of limited quality for PCR
  • 13% of samples were not evaluable on the tissue microarray format because of inadequate pepsin digestion or loss of tissue cores
  • The BCL6 break-apart probe did not detect inversions and deletions involving band 3q27 that chromosome banding found

Topics

Medical terms in this study, explained in our Lexicon: Fluorescence In Situ Hybridization (FISH), Translocation, Mesothelioma, Paraffin Block.

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