Abstract P3-05-05: HER2 Expression and Gene copy analysis by Immunofluorescence and Fluorescence in situ Hybridization, on a Single formalin-fixed paraffin-embedded tissue section
Authors: Ha T , Seppo A , Ginty F , Kenny K , Henderson D , Kyshtoobayeva A , Gerdes M , Larriera A , Liu X , Corwin A , Zingelewicz S , Lazare M , Jun N , Kyshtoobayeva A , Chow C , Al-Kofahi Y , Hollman D , Bloom K 
Published: August 2014

Abstract
Introduction: Breast cancer is the most common cancer for women worldwide. HER2 expression and gene copy number are important when determining eligibility for adjuvant therapy and/or chemotherapy medications. One challenging issue for breast cancer testing is intratumoral heterogeneity of HER2 gene amplification. Intratumoral heterogeneity can make it difficult to localize target cells of interest. Serial tissue sections used for independent H&E, IHC and FISH stains also increase the difficulty to localize targets due to cellular truncation. We have developed a system to assess both HER2 expression and gene copy number on the same cell.
Method: Immunofluorescence (IF) and Fluorescence in situ Hybridization (FISH) were performed on tissue sections from 19 patients with invasive ductal breast carcinoma. Cases were selected based on prior HER2 FISH results (HER2:Chromosome 17 = ratio) representing unamplified (&lt;2.0), amplified (≥2.0) and equivocal (1.8–2.2). Samples were collected from June 2011 – February 2012. Tissue sections were cut at 4uM from formalin-fixed paraffin-embedded tissue blocks. Slides were stained with antibodies for HER2 (Clone #D8F12, Cell Signaling, Danvers, MA), cytokeratin (Clone #AE1, eBioscience, San Diego, CA) and Pan cytokeratin (Clone #PCK-26, Sigma-Aldrich, St. Louis, MO). The whole tissue imaging was performed on the In-Cell (GE Healthcare, Chalfont St. Giles, UK) at 10X. Proprietary software developed by GRC (GE Global Research, Niskayuna, NY) controlled the hardware and performed numerous algorithmic functions. Regions of Interest (ROI) were selected by a pathologist on a whole tissue image and coordinates were recorded by the software. The slides were then imaged at 40x using the previously recorded ROI's. The same slides were stained with the PathVysion HER2/CEP17 FISH kit (Abbott Molecular, Des Plaines, IL). Slides were registered to the previous IF scan using recorded coordinates and tissue morphology recognition algorithms. The sections were imaged for FISH at 40X using the previous ROI selections. Cases were assessed for successful protein and genetic expression using proprietary visualization tools for combined analysis.
Results: We evaluated a total of 22 breast cancer cases with 19 cases detecting both protein and gene expression. Of the three cases that could not be evaluated the rationale is as follows: tissue damage incurred during imaging, insufficient focus during the FISH imaging portion, and poor signal to noise of the FISH dots.
Conclusion: The reported incidence of intratumoral HER2 amplification heterogeneity is as high as 30%. The challenges associated with tumor heterogeneity may benefit from a standardize analysis method. Using integrated images generated by this system, pathologist is able to select the appropriate cells for HER2 copy number enumeration based on the expression level of HER2 protein, in the same cell, allowing rapid identification of intratumoral heterogeneity.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-05.