Abstract P1-01-13: Does HER2 immunohistochemistry-guided targeted FISH analysis help identify intratumoral heterogeneity in breast cancer?
Authors: Gulbahce HE , Factor RE , Geiersbach KB , Downs-Kelly E 
Published: May 2016

Introduction: The updated 2013 ASCO/CAP HER2 guidelines proposed revised scoring with the aim of capturing all patients eligible for targeted therapy. The current guidelines also address intratumoral heterogeneity using immunohistochemistry (IHC) as a platform to identify and target heterogeneously staining areas for FISH analysis. We wanted to determine if identifying the most intense areas of staining in IHC equivocal / 2+ cases would help identify genetic heterogeneity and potentially more FISH amplified patients.

Materials and Methods: Our lab offers HER2 immunohistochemistry by HercepTest™ (Dako) or 4B5 (Ventana), and dual probe FISH (Abbot Molecular or Dako IQ). We offer HER2 testing as IHC or FISH only, and also as a reflex FISH following equivocal (2+) IHC. HER2 tests performed and interpreted after 10/2013, following the implementation of the ASCO/CAP 2013 guidelines, to 5/2015 were included in this study. Both IHC and FISH were manually read following ASCO/CAP 2013 guidelines by one and two observers respectively. Equivocal (2+) cases with heterogeneous staining showing areas with ≥ 10% weak or moderate circumferential membrane staining were circled by a pathologist and reflexed to HER2 FISH. In those cases where samples were received for FISH testing only, the pathologist circled the entire area of invasive tumor on H&E slides. All circled tumor areas were evaluated before signal enumeration of the FISH probes.

Results: 1805 HER2 FISH test requests received in our laboratory during the study period had interpretable FISH results. 1210 of these cases did not have prior HER2 IHC testing performed in our laboratory ("FISH only"), 595 had reflex FISH testing following equivocal (2+) IHC ("IHC2+/reflex FISH"). Amplification rates between the "IHC2+/reflex FISH" group of patients with targeted areas for FISH and the "FISH only" group where the entire invasive carcinoma was marked on H&E slide and scored were similar (16.3% and 20% respectively). 10/595 (1.7%) "IHC2+/reflex FISH", and 15/1210 (1.4%) "FISH only" groups showed genetic heterogeneity by FISH with discrete population of amplified tumor cells respectively (p=0.4)

 FISH Testing Utilizing Entire Tumor Area * n=1210FISH Testing Utilizing Most Intense Staining Areas (following equivocal IHC) n=595Genetic Heterogeneity15 (1.4%)10 (1.7%)Amplified242 (20.0%)97 (16.3%)*includes equivocal IHC, and FISH as first line of testing

Conclusion: The updated HER2 ASCO/CAP guidelines recommend that intratumoral heterogeneity for HER2 may be easier to detect with IHC which can be used to guide FISH enumeration. In our study, identifying areas of more intense staining on IHC slides for targeting FISH analysis did not result in a difference in identification of genetic heterogeneity.

Citation Format: Gulbahce HE, Factor RE, Geiersbach KB, Downs-Kelly E. Does HER2 immunohistochemistry-guided targeted FISH analysis help identify intratumoral heterogeneity in breast cancer?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-13.