Original Article

Analytical Validation and Interobserver Reproducibility of EnzMet GenePro

A Second-Generation Bright-Field Metallography Assay for Concomitant Detection of HER2 Gene Status and Protein Expression in Invasive Carcinoma of the Breast

Downs-Kelly, Erinn DO*; Pettay, James MT (ASCP)*; Hicks, David MD*; Skacel, Marek MD*; Yoder, Brian DO, PhD*; Rybicki, Lisa MS†; Myles, Jonathan MD*; Sreenan, Joseph MD‡; Roche, Patrick PhD§; Powell, Richard PhD∥; Hainfeld, James PhD∥; Grogan, Thomas MD§; Tubbs, Raymond DO*

Author Information

From the *Departments of Anatomical and Clinical Pathology, †Biostatistics and Epidemiology, Cleveland Clinic Foundation and the Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH; ‡St. Rita's Medical Center, Lima, OH; §Ventana Medical Systems International, Tucson, AZ; and ∥Nanoprobes Incorporated, Yaphank, NY.

Supported by grant no. 2R42CA83618-02 and 5R42 CA083618-03 from the National Cancer Institute (NIH) to Nanoprobes, Inc and the Cleveland Clinic Lerner College of Medicine; and by grant number 1R43 GM064257-01 from the National Institute of General Medical Sciences (NIH) to Nanoprobes, Inc and the Cleveland Clinic Lerner College of Medicine. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or the National Institute of General Medical Sciences.

Reprints: Raymond R. Tubbs, DO, 9500 Euclid Ave (L11), Cleveland, OH 44195 (e-mail: [email protected]).

The American Journal of Surgical Pathology 29(11):p 1505-1511, November 2005. | DOI: 10.1097/01.pas.0000172294.67409.4f

Abstract

Fluorescence in situ hybridization (FISH) has both excellent sensitivity and specificity in detecting HER2 gene amplification in invasive breast carcinoma. FISH has not been widely implemented in clinical practice because of reagent costs and the special instrumentation and expertise required to perform and integrate the assay. Immunohistochemistry (IHC) for HER2 protein is widely used, but false-positive and false-negative results are problematic. We developed a bright-field assay to visualize HER2 gene amplification and concomitant HER2 protein expression (EnzMet GenePro). This assay detects HER2 gene amplification via deposition of metallic silver by enzyme metallography™ (EnzMet™, Nanoprobes, Yaphank, NY) combined with HER2 protein detection by IHC using alkaline phosphatase and fast red K substrate visualization (CB11;Ventana, Tucson, AZ). The assay was performed on 94 invasive breast carcinomas, for which FISH (PathVysion™, Vysis, Downer's Grove, IL), conventional IHC (CB11), and enzyme metallography (EnzMet™) results were known. The EnzMet™ component of the assay was scored as either HER2 gene amplified, polysomic, or nonamplified. The IHC component was scored using the conventional FDA scale of 0 to 3+. Concordance of the EnzMet component of the assay versus FISH was assessed and showed an excellent correlation (Pearson coefficient of 0.95; P < 0.001). The combination of gene and protein detection (EnzMet GenePro) displayed a specificity of 100% and an accuracy of 92.6% (95% confidence interval 85.3-97.0), facilitated recognition of gene/protein discordances, and allowed for efficient interpretation of the slide by conventional light microscopy. The interobserver kappa for each component was excellent (IHC, κ = 0.94; and EnzMet™, κ = 0.96). EnzMet is the first bright-field ISH assay in our experience that routinely and nonambiguously detects endogenous HER2 signals, essential for a reliable clinical HER2 assay, and in combination with HER2 protein enables improved diagnosis in borderline cases.

© 2005 Lippincott Williams & Wilkins, Inc.

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