Clinical Chemistry
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Clinical Chemistry 54: 1831-1839, 2008. First published September 11, 2008; 10.1373/clinchem.2008.107607
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(Clinical Chemistry. 2008;54:1831-1839.)
© 2008 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Liquid-Based Fluorescence In Situ Hybridization Assay for Detection of ERBB2 Gene Amplification in Patients with Breast Cancer3

Chen-Hsiung Yeh1, William A. Whitmire, III1 and Maher Albitar1,a

1 Department of Hematopathology, Quest Diagnostics Nichols Institute, San Juan Capistrano, CA.

aAddress correspondence to this author at: Department of Hematopathology, Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92675-2042. Fax 949-728-7781; e-mail Maher.X.Albitar{at}questdiagnostics.com.

Background: Current reference methods for evaluating gene amplification and expression of ERBB2 (also known as HER-2)—cell-based fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC)—are subjective and influenced by methods of tissue preparation and fixation. We developed and evaluated a novel, quantitative liquid-based FISH (L-FISH) assay that uses flow cytometry to detect ERBB2 gene amplification in breast cancer patients.

Methods: DNA was extracted from serum or tissue, biotinylated, hybridized to differentially labeled probes for ERBB2 and a chromosome 17–specific single-copy sequence (17-SSC), and immobilized to streptavidin-coated microspheres. The ERBB2/17-SSC signal ratio measured by flow cytometry was used to evaluate ERBB2 amplification. We used L-FISH to test 122 stored formalin-fixed, paraffin-embedded (FFPE) tissue samples and 22 serum samples from randomly selected breast cancer patients; results were compared with those obtained with conventional FISH and IHC.

Results: The inter- and intraassay imprecisions were 3.7%–18.9% for FFPE tissue and 2.8%–6.3% for serum. Overall, L-FISH analyses of FFPE tissues demonstrated 84.4% concordance with results obtained with conventional FISH (P < 0.001) and 78.8% concordance with IHC results (P < 0.001). L-FISH analyses of serum samples showed 91% concordance with tissue-based IHC/FISH results (P = 0.038).

Conclusions: Our data indicate that this PCR-free L-FISH method can be used to evaluate ERBB2 amplification in both cell-containing (paraffin-embedded tissue) and cell-free (serum) samples. This approach provides more objective results and is amenable to automation and quantitative measurement.






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Copyright © 2008 by the American Association for Clinical Chemistry.