Abstract P5-10-03: Audit of the accuracy of immunohistochemical (IHC) testing of HER2 negative status of breast cancer in the United Kingdom
Authors: Jasani Bharat , Campbell Fiona , Jones Phillapa , Gilbert Jane , Dowd James , Miller Keith , Ibrahim Merdol , Ellis Ian , Hurley Emma , Falzon Mary , Peter Barrett-Lee , Starczynski Jane 
Published: May 2015

Abstract
Background: The analysis of the level and distribution of HER2 protein expressed by cancer cells (HER2 status) is of great clinical value in the management of breast cancer patients both for the determination of the prognosis of disease and for identification of those patients who are eligible for anti-HER2 therapy. Accurate assessment of the HER2 status is essential for identifying patients which will benefit from HER2 targeted therapy. HER2 status in the UK is established using a two tier strategy with IHC as the initial test and subsequent reflex of equivocal results to in situ hybridization (ISH). IHC staining of the HER2 protein is graded as 0, 1+, 2+ or 3+ dependent upon the intensity of staining, cellular localisation and the percentage
of cells positive in accordance with CAP/ASCO and UK guidelines. HER2 3+ cases are considered as positive, with HER2 2+ cases (equivocal) retested by ISH to ascertain the gene amplification status. Cases that are scored as 0 and 1+ by IHC have no additional testing and are classed as negative. The literature indicates that a subset of these IHC negative cases show HER2 gene amplification by FISH (range 1.1-11.5%). The aim of this audit is to evaluate the discordance rate of HER2 IHC negative,
FISH positive breast cancer in the UK, with a secondary objective to resolve if this is related to the choice of antibody and assay platform used.
Materials and methods: This audit selected a total of 600 sequential cases reported as HER2 negative on IHC, from three UK reference centres receiving cases from 29 different hospitals. The cases were given a unique identifying number and annonymised. Each of the three centres used a different IHC method for frontline HER2 testing with centre one using HercepTestTM (DAKO), centre two Pathway 4B5 (Roche), and centre three, Oracle (Leica Microsystems). HER2 gene amplification status was determined using dual colour FISH analysis, PathVysion (ABBOTT) fluorescence ISH (FISH)
in a single centre to provide standardised methodology and assessment. HER2 was classed as amplified when the HER2/CEP 17 ratio was two or greater in accordance with UK guidelines. All cases which showed discordance between IHC and FISH were re-tested with each of the HER2 IHC platforms to discover whether these are truly discordant results or if the discrepancy is a consequence of the choice of antibody.
Results: 16/600 (2.8%) unequivocal HER2 gene amplification (mean ratio &gt;2.0) whilst 8/600 (1.2%) had borderline amplification status(mean ratio = or &lt;2.0). The overall assay specific discordance rates were 3.0% (HercepTest), 2.5% (4B5) and 3.0% (Oracle), respectively.
Conclusion: The observed level of discordance is well within the range of discordance rates reported by previous studies. The discrepancies could be due to inadequate quality fixation and/or inadequate sensitivity of the assay platforms used, or under scoring. A detailed analysis of possible assay related source of discrepancy is currently underway by repeating the analyses of the 24 discordant cases using like for like three assay platforms at an independent expert centre.
Citation Format: Bharat Jasani, Fiona Campbell, Phillapa Jones, Jane Gilbert, James Dowd, Keith Miller, Merdol Ibrahim, Ian Ellis, Emma Hurley, Mary Falzon, Barrett-Lee Peter, Jane Starczynski. Audit of the accuracy of immunohistochemical (IHC) testing of HER2 negative status of breast cancer in the United Kingdom [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-03.