High Concordance Between Herceptest Immunohistochemistry and ERBB2 Fluorescence in Situ Hybridization Before and After Implementation of American Society of Clinical Oncology/College of American Pathology 2007 Guidelines

Maria E Vergara-Lluri; Neda A Moatamed; Elizabeth Hong; Sophia K Apple


Mod Pathol. 2012;25(10):1326-1332. 

In This Article

Materials and Methods


Formalin-fixed, paraffin-embedded human breast cancer tissue specimens from 1437 patients with invasive breast carcinoma were used in the study. Both immunohistochemical analysis for HER2 protein and fluorescence in-situ hybridization for ERBB2 gene were performed on all specimens from our facility at David Geffen University of California at Los Angeles (UCLA) Medical Center from years 2003 through 2010 (n=1437). All samples were collected from 2003–2010, with testing results available for each sample. Optimal tissue handling requirements (eg, time to fixation) were followed and recorded, especially after publication of the ASCO/CAP guidelines, on 1 January 2008. To decrease pre-analytic variables in testing, optimal internal validation procedures, internal quality assurance procedures, external proficiency assessment, laboratory accreditation, and immunohistochemistry and fluorescence in-situ hybridization testing requirements were met in accordance with the ASCO/CAP guideline recommendations. A retrospective review of data was performed. No further testing has been done in relation to the study. An application to the Internal Scientific Peer Review Committee was submitted and approved. Subsequently, the study was conducted according to Office of Human Research Protection Program, and was approved by the University of California at Los Angeles Institutional Review Board.

Immunohistochemical Analysis

The US FDA-approved HercepTest was performed using DAKO A0485 polyclonal antibody kit (DAKO Corp, Carpenteria, CA, USA). Cases from 2003–2007 (n=1016) were scored by US Food and Drug Administration (US FDA) guidelines before publication of ASCO/CAP guidelines, with immunohistochemistry of 3+ cases staining >10% of tumor cells. The new ASCO/CAP guidelines were implemented and scored accordingly for cases from 2008–2010 (n=421), into three categories: negative for HER2 protein overexpression (scores 0 and 1+), indeterminate (2+), and positive for HER2 protein overexpression (3+).

Score 0: No staining is observed in invasive tumor cells.

Score 1: Weak, incomplete membrane staining in any proportion of invasive tumor cells, or weak, complete membrane staining in less than 10% of cells.

Score 2: Complete membrane staining that is non-uniform or weak but with obvious circumferential distribution in at least 10% of cells, or intense complete membrane staining in 30% or less of tumor cells.

Score 3: Uniform intense membrane staining of more than 30% of invasive tumor cells.

Fluorescence In-Situ Hybridization Analysis

Fluorescence in-situ hybridization was performed using the US FDA-approved PathVysion HER-2 DNA Probe Kit (PathVysion Kit), which is designed to detect amplification of the ERBB2 gene via fluorescence in-situ hybridization in formalin-fixed, paraffin-embedded human breast cancer tissue specimens. Fluorescence in-situ hybridization analysis with VYSIS dual-color probes specific for chromosome 17 centromere and the ERBB2 gene (17q11.2) was performed and examined by two independent technologists, and signed out by clinical pathologists (non-surgical anatomic pathologists) at the UCLA Cytogenetics Laboratory. Slides containing 4 μm sections were submitted for fluorescence in-situ hybridization analysis. For each slide, based on the corresponding hematoxylin and eosin slide, the invasive tumor area(s) was circled with a secureline marker. Areas containing ductal carcinoma in-situ or normal tissue were excluded from fluorescence in-situ hybridization testing, as HER2 protein overexpression and ERBB2 gene amplification are seen more frequently in ductal carcinoma in-situ (50–60%) than in invasive carcinoma of the breast (25–30%).[15] Slides were baked overnight at 60 °C and pretreated using the VP2000 tissue processor as per manufacturer's protocol (Abbott Molecular, Abbott Park, IL, USA). Amplification of the ERBB2 gene was detected by using the PathVysion Kit; the instructions in the package insert were followed for the hybridization, post-hybridization washing, and analysis steps (Abbott Molecular).

Cases from 2003–2007 (n=1016) were scored according to US FDA guidelines for HER2 immunohistochemistry and using ERBB2 gene fluorescence in-situ hybridization amplification cutoff value of 2.0 ( Table 1 ). The new ASCO/CAP guidelines were implemented and scored accordingly for cases from 2008–2010 (n=421) ( Table 2 ). ERBB2 gene amplification was defined as ERBB2/CEP17 ratio of ≥2.2. For the purposes of this study, no amplification was defined as an ERBB2/CEP17 ratio of <2.2.

A comparison of the data between 2003–2007 using US FDA Guidelines and 2008–2010 using ASCO/CAP Guidelines is shown ( Table 3 ).

Statistical Analysis

The calculation of concordance rates and k between the HER2 immunohistochemical analysis and ERBB2 fluorescence in-situ hybridization assays, and χ 2-tests, were performed on SPS software. The results of discordant cases were included in the data analysis before trouble shooting.