HER2 is a prognostic and predictive marker for breast cancer patients and its expression is routinely evaluated by immunochemistry (IHC). Scoring of IHC slides is prone to operator subjectivity and equivocal results make selection of appropriate therapy difficult, ultimately affecting patient outcomes. We describe development and validation of a reproducible quantitative immunofluorescence assay to accurately assess HER-2 levels in Formalin-Fixed Paraffin-Embedded (FFPE) breast cancer specimens by AQUA Technology that avoids the ambiguity of equivocal results and enables critical treatment decisions.

Methods: A tissue microarray (n=80) composed of breast cancer cases with known IHC and fluorescence in situ hybridization (FISH) status was used to characterize and optimize assay performance for three HER-2 antibody clones; A0485, CB11, and SP3. Eight dilutions of each antibody were tested with four different antigen retrieval conditions. A total of 108 assay conditions were evaluated by receiver operator characteristic (ROC) analysis for sensitivity and specificity. Equal weight was given to sensitivity and specificity to select the most robust assay. The top six assay conditions were then assessed using 45 whole tissue breast cancer specimens to identify one condition with highest sensitivity and specificity. The selected assay condition was evaluated on an additional 90 breast cancer specimens (training set) annotated for IHC and FISH scores to determine the binary cut point for the HER-2 AQUA assay®, the cut point was then confirmed on a validation set composed of 90 independent breast cancer specimens. The final assay was analytically validated in accordance with College of American Pathologists (CAP) guidelines utilizing over 120 independent breast cancer specimens.

Results: Based on an evaluation of over 400 breast cancer specimens with nearly equal distribution of 0, 1+, 2+ and 3+ cases, HER-2 antibody clone, SP3, which recognizes the extracellular domain of the receptor, clearly segregated HER2 positive specimens from HER2 negative breast cancer cases.

Conclusion: The availability of a highly reproducible quantitative binary test facilitates rapid treatment decisions with appropriate HER-2 targeting biologics on the market.

Citation Format: Lakshmi Krupa Chandrasekaran, Jennifer Bordeaux, Sue Beruti, Naveen Dakappagari, Mike Nerenberg, Jelveh Lameh, Armin Graber, David Rimm, Bruce Robbins, Nagesh Rao. Development of a binary diagnostic immunofluorescence assay by AQUA® technology for accurate detection of HER-2 levels in breast cancer specimens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2843. doi:10.1158/1538-7445.AM2014-2843