Abstract P1-02-03: Clinical utility of combined brightfield HER2 gene-protein assay in IHC equivocal cases
Authors: Bhargava R , Dabbs DJ 
Published: August 2014

Abstract
The combined gene protein assay (GPA) for HER2 (Ventana Medical Systems Inc, Tucson AZ) is a newly developed technique where both HER2 gene and protein can be simultaneously assessed on a single slide using routine bright field microscopy. This is made possible by combining HER2 immunohistochemistry (using clone 4B5 from Ventana) with the HER2 Dual in-situ hybridization (DISH) DNA probe cocktail. The main goal of the study was to compare this new GPA methodology with standard fluorescence in situ hybridization (FISH) on the most challenging group of breast cancers, i.e. HER2 immunohistochemical (IHC) 2+ cases.
This validation study included whole tissue sections of 36 invasive breast cancer cases with equivocal HER2 IHC results (IHC score 2+) on clinical testing. FISH and GPA were performed on the same tumor blocks. The GPA assay staining results were enumerated by counting at least 20 nuclei by a pathologist blinded to FISH results. Similar to FISH, areas with stronger IHC staining were counted in heterogeneous cases. For both FISH and GPA, HER2 amplification was defined as HER2/CEP17 ratio ≥ 2.0. Cases with HER2/CEP17 ratio &lt;2.0 were considered as non-amplified. In addition, 25 known HER2 IHC 3+ invasive carcinoma cases (represented on a tissue microarray) and 22 known IHC negative (scores 0 and 1+ also represented on tissue microarray) were also evaluated by GPA.
GPA was successful in all cases in the first attempt. All previously tested IHC 2+ cases were again scored as 2+ on GPA. The agreement for IHC 2+ cases between FISH and GPA with respect to HER2 gene status was 94.4% (see table). The 2 discordant cases were amplified by GPA and not amplified by FISH. The HER2 to CEP17 ratio on these 2 cases by GPA was 2.0 and 2.34 with average HER2 copies/cell of 5.7 and 5.5 respectively. For the 36 IHC 2+ cases, the mean HER2 copies per cell for FISH were 3.09 (std dev: 1.8; range: 1.9 to 11.07) versus 3.52 (std dev: 1.8; range: 1.8 to 9.95) for GPA. The mean CEP17 signals per cell were 2.24 (std dev: 0.6; range: 1.65 to 4.2) for FISH versus 2.3 (std dev: 0.6; range: 1.6 to 3.5) for GPA. The mean for HER2:CEP17 ratio was 1.34 (std dev: 0.6; range: 1.03 to 4.32) by FISH and 1.50 (std dev: 0.7; range: 1.02 to 4.72) by GPA.
GPA versus FISH for IHC 2+ cases GPA amplifiedGPA not amplifiedTotalFISH amplified202FISH not amplified23234Total43236FISH: Fluorescence in-situ hybridization; GPA: Gene protien assay
All 25 (100%) of IHC 3+ cases showed clusters of HER2 gene consistent with HER2 amplification by GPA. These 25 cases also showed strong membranous (“chicken wire”) reactivity consistent with 3+ IHC score. Similarly, the 22 known IHC negative cases on the TMA, were again IHC negative, and lacked HER2 gene amplification by GPA.
The HER2 GPA shows excellent concordance with both HER2 IHC assay and FISH assay. It performs accurately not only for unequivocally positive and negative cases but also on the most challenging (IHC 2+) breast cancer cases. This new assay combines the advantages of both IHC and FISH, i.e. accuracy similar to FISH for IHC equivocal cases, faster turn around time (less than 24 hours) like IHC and evaluation under bright-field microscope where the tissue morphology is well preserved and heterogeneity is better appreciated.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-03.