Abstract PO4-26-05: Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution
Authors: Xu Xiaocao , Ambaye Abiy , Landry Kara , Kaufman Peter 
Published: May 2024

Abstract
Background:
HER-2 critically guides therapies and is prognostic in breast cancer (BC). Current ASCO/CAP guidelines for immunohistochemistry (IHC) define 3+ as positive, 0 and 1+ as negative, and 2+ as equivocal, with additional testing recommended. Current guidelines for in situ hybridization (ISH) define a HER-2/CEP17 ratio of ≥2.0, with an average HER-2 copy number of ≥4.0 signals/cell as ISH positive. With &lt; 4.0 copies HER-2/cell, and for various other ISH groups that are borderline, further specific additional testing is recommended. Data suggest that HER-2 copy number does have clinical relevance in HER-2+ BC, however most clinical trials evaluating HER-2 targeted therapeutics have defined HER-2+ as specifically a HER-2/CEP17 ratio ≥2.0. Limited studies have explored HER-2 copy number and its clinical implication in the HER-2 1+, and 2+ ISH negative population, recently described as HER-2 low. Further, the guidelines for HER-2 testing have changed twice over the past decade, and little is known about the impact of these changes.
Design:
From 2001- March 2023, all new invasive BC cases tested at University of Vermont (UVM) for HER-2 with both IHC and ISH were analyzed. All testing was performed on formalin-fixed paraffin-embedded tissue, using the HercepTest kit (Dako Corp, Carpinteria, California) (2001-2012) 4B5 Rabbit Monoclonal Antibody (Ventana) (2013-2023) for IHC and the INFORM dual ISH DNA probe (Ventana) for CISH. Interpretation was performed using published criteria at the time of original testing, and all cases with both IHC and CISH testing were independently analyzed by two experienced reviewers.
Results:
2734 cases had dual testing with IHC and CISH. 2407 patients (88%) had a HER-2/CEP17 ratio &lt; 2.0, of these, 6.4% had a HER-2 copy number ≥4 and &lt; 6. Only 12% of cases overall had a ratio ≥ 2.0. In IHC 0 and 1+ cases with HER-2/CEP17 ratio of &lt; 2, 0.4% and 4.3% have excess HER-2 copies/cell (defined as ≥ 4), respectively, compared to 13.8% for IHC 2+. For IHC 2+ cases, only 13.6% overall demonstrate amplification, with however, again, an additional 13.8% having excess copy number. Interestingly, 9.6% of the IHC 3+ cases had a ratio of &lt; 2.0, however 50% of those demonstrate HER-2 amplification. Overall, 6.4% of HER-2 non-amplified cases with a ratio &lt; 2.0 have an excess HER-2 copy number. In our full institutional pathology database, 11.2% of cases are HER2+, by either IHC or ISH. Lastly, 43% of cases overall, at UVM, from 2018 – March 2023 would fall into the current classification as HER-2 low (IHC 1+, or IHC 2+ and ISH negative), as well.
Additionally, we have evaluated the change in IHC and ISH results that occurred with the changes in the ASCO/CAP guidelines in both 2013 and 2018, further data will be available for presentation.
Conclusion:
In this large single institutional analysis of dual HER-2 testing, only 12% of cases demonstrate HER-2 amplification, and overall in our institutional pathology database, from 2001 – March 2023, 11.2% of cases are HER-2+ by either IHC or ISH. In our dual tested cohort, only 13.6% of IHC 2+ cases demonstrate amplification, and 13.8% are non-amplified, but have excess HER-2 copy number. We demonstrate a numerical increase in the rate of amplification, and copy number in non-amplified cases, associated with IHC score. In all cases tested with IHC at UVM from 2018 – March 2023, we find 43% of cases meet the current criteria for HER-2 low. Finally, the ASCO/CAP guideline changes from 2013 to 2018 had modest impact on the overall percentage of HER-2+ cases in our series, further data on this impact will be available for presentation.
Table. Simultaneously tested IHC and CISH data for new invasive breast cancer cases
Citation Format: Xiaocao Xu, Abiy Ambaye, Kara Landry, Peter Kaufman. Dual HER2 Testing With Both Immunohistochemistry (IHC) and ISH (In Situ Hybridization): Long Term Analysis of Dual Testing in a Single Academic Institution [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-05.