Abstract P4-09-22: Quantitative immuno-fluorescent evaluation of Her2 expression levels in a prospectively collected cohort of breast cancer cases: Comparison to conventional IHC scoring and FISH

Background: According to the 2013 guidelines breast cancers are defined as Her2 positive if there is evidence of protein expression in at least 10% of tumor cells by IHC and/or gene amplification by FISH. Nevertheless, there are still IHC 2+ and FISH equivocal breast cancers resulting in repeat testing. It is also known that not all Her2 positive breast cancers respond to Trastuzumab, while up to 8% of Her2 "negative" classified patients benefit from Her2 targeting regimens. Toward the goal of generating a more accurate test, we report in situ quantification of Her2 protein levels on a prospectively collected cohort of breast cancers and comparison to conventional IHC and FISH evaluation. Materials and Methods: A prospectively designed study was initiated at Yale University, comparing quantitative, in situ measurement of Her2 protein levels with conventional IHC and FISH evaluation. All breast cancer specimens were analyzed by IHC and FISH in our routine clinical laboratory, read and signed out by the breast pathologists. Serial sections were then stained and quantified for Her2 expression levels using the AQUA method of quantitative immunofluorescence (QIF). Data for all assays were obtained on 120 samples over a period of 6 months. Staining was performed using the DAKO Herceptest and the Epitomics EP3 Her2 antibody for IHC, the DAKO rabbit polyclonal antibody for QIF. The 30 highest cases were then retested for QIF and IHC using the Biocare c-erbB-2 [CB11] antibody. Each staining run included an index tissue microarray (TMA) consisting of 80 cases, cell lines and normal tissue for quality control, assay reproducibility and threshold definition of AQUA scores correlated to HER2 over expression and amplification. Results: Out of 120 specimens analyzed for HER2, 13 were diagnosed as IHC 2+/3+, FISH amplified, 1 case had an equivocal score, 14 cases were IHC 2+/non amplified, 2 cases IHC 1+/FISH amplified and 89 specimens IHC 0/1+ non amplified. The continuous AQUA scores for Her2 expression of the samples significantly correlate with traditional clinical Her2 scoring. However, 5 IHC 0/1+, non amplified cases revealed high AQUA scores in the range of HER2 overexpression/amplification. Repeat testing of these by both QIF and IHC showed reproducibility of the results. AQUA scores of one IHC 3+/amplified sample were lower than the threshold of HER2 overexpression/amplification. Conclusions: QIF measurement of HER2 protein levels in a prospectively collected cohort of 120 breast cancer specimens reveals significant association between continuous HER2 protein levels and the ordinal conventional scoring system. However, five discordant cases that were above the threshold for HER2 protein by QIF, were classified as negative by conventional methods. Given the accuracy and reproducibility of the QIF test, it raises the possibility that some of these patients might benefit from HER2 targeted therapy. In summary, while continuous scoring of HER2 protein correlates well with conventional methods, it identifies a subset of patients that are discordant with current methods. Further comparative studies in a patient cohort with response to targeted therapy need to be evaluated. Citation Format: Neumeister VM, Yan SS, McGuire JA, Carvajal DE, Prasad ML, Rimm DL. Quantitative immuno-fluorescent evaluation of Her2 expression levels in a prospectively collected cohort of breast cancer cases: Comparison to conventional IHC scoring and FISH. [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 P4-09-22.