Abstract PS5-11: Prospective HER-2 testing using simultaneous immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH): Experience of dual testing in a single academic institution
Authors: Landry Kara K , Ambaye Abiy B , Weaver Donald L , Kaufman Peter A 
Published: February 2021

Abstract
Background: HER-2 testing informs prognosis and critically guides therapy in breast cancer. Current guidelines for immunohistochemistry (IHC) testing define 3+ as positive, 0 and 1+ as negative, and 2+ as equivocal with additional testing recommended. Guidelines for in situ hybridization (ISH) testing are more complex and generally define a HER-2/CEP17 ratio of ≥2.0 with average HER-2 copy number of ≥4.0 copies/cell as HER-2 positive. Further, HER-2 copy number between 4 and 6 per cell, with a ratio of &lt;2.0, is considered equivocal while &gt;6 per cell is frequently confirmed positive. Limited data suggest that an elevated HER-2 copy number alone has clinical relevance, however most clinical trials addressing HER-2 targeted therapeutics define HER-2 positive based on HER-2/CEP17 ≥2.0.
Design: From 2013-2016, all new invasive breast cancer cases diagnosed at the University of Vermont Medical Center were prospectively tested for HER-2 using both IHC and chromogenic in situ hybridization (CISH) simultaneously. All testing was performed on formalin-fixed paraffin-embedded tissue, using the 4B5 Rabbit Monoclonal Antibody (Ventana) for IHC and the INFORM dual ISH DNA probe (Ventana) for CISH. Interpretation was performed using published criteria, and all cases were independently analyzed by two experienced reviewers.
Results: 1906 patients underwent HER-2 testing by both IHC and CISH. 1671 (87.7%) patients had a HER-2/CEP17 ratio of &lt;2.0. Of patients considered to be non-amplified by the CISH ratio, 55 (3.2%) had a HER-2 copy number &gt;4. In IHC 0 and 1+ cases with HER-2/CEP17 ratio of &lt;2, 0.4% and 2.3% have equivocal and excess HER-2 copies/cell, respectively, compared to 10.8% for IHC 2+. Additionally, 17 (8.6%) of the IHC 3+ cases had a CISH ratio of &lt;2, of which 41.2% had &gt;4 HER-2 copies/cell (see table).
Conclusion: To our knowledge, this is the largest study to date that has examined the concurrent use of both IHC and ISH HER-2 assays prospectively in a real-time clinical setting. In the IHC 2+ cohort, only 12.5% were amplified by ISH testing. Interestingly, of those in the IHC 2+ cohort not amplified by CISH, 10.8% had an equivocal or excess HER-2 copy number. Further, while based on a very small sample size, 41.2% of IHC 3+ patients with a CISH ratio of &lt;2.0 had an equivocal or excess HER-2 average copy number. Finally, data on the clinical outcome and therapeutic benefit of HER-2 targeted therapy in patients with non-amplified ISH testing and equivocal or excess HER-2 copy number remains limited, and warrants further evaluation.
Table. Simultaneously tested IHC and CISH data for new invasive breast cancer casesIHCCISH ratio ≥2.0CISH ratio &lt;2.0HER-2 copies/cell (CISH ratio &lt;2.0 cohort)CISH ratio &lt;2.0 with ≥4 HER-2 copies/cellIHCnn (%)n (%)&lt;4 (n)4-6 (n)&gt;6 (n)05010 (0.0)501 (100.0)499200.4%1+94221 (2.2)921 (97.8)8992012.3%2+26533 (12.5)232 (87.5)20625010.8%3+198181 (91.4)17 (8.6)106141.2%Total1906235 (12.3)1671 (87.7)16145323.2%
Citation Format: Kara K Landry, Abiy B Ambaye, Donald L Weaver, Peter A Kaufman. Prospective HER-2 testing using simultaneous immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH): Experience of dual testing in a single academic institution [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-11.