Abstract A41: Validated immunohistochemistry assay for measuring total Aurora A kinase protein levels in FFPE human tumors.
Authors: Danaee Hadi , Leroy Patrick , Lichter David I. , Ecsedy Jeffrey , Mulligan George , Trepicchio William L. 
Published: June 2012

Abstract
Aurora A kinase (AURKA) is a serine threonine kinase that plays a critical role in regulating mitotic progression during the cell cycle. Numerous reports have shown that AURKA is frequently activated in a multitude of tumor indications. The activation of AURKA can occur either at the chromosome level with gene amplification and/or at the protein level with overexpression. The overexpression of AURKA in tumors has been linked to the etiology of some cancers and to a worsened prognosis in patients. Detection of AURKA protein in these previous studies by immunohistochemistry (IHC) on human tumors has not been done using a standardized validated assay. This presents a challenge in comparing AURKA protein expression data across numerous studies. Published reports have noted AURKA protein levels in tumors ranging from 15% to as high as 94% depending on the tumor type. Evaluation of the staining pattern in these reports raised the question of whether this is true AURKA staining that is being detected, and whether it would be anticipated that a mitotic kinase with predominant expression during mitosis would be this prevalent in these tissues.
In order to better understand the protein expression level of total AURKA in human tumors, we developed an IHC assay for AURKA that could be used on formalin fixed paraffin embedded (FFPE) tissue. Initially, 4 different AURKA primary antibodies (Epitomics, Leica Microsystems (Novocastra), CST, and Becton Dickenson) were evaluated by Western blot analysis on lysates from asynchronous and nocadazole treated cells to estimate the specificity of each antibody. Matched frozen and FFPE human tumors were then measured by western blot and IHC respectively for AURKA protein levels to establish concordance between the two methods. Testing on additional human FFPE tumors and 5 different tissue micro arrays (TMA) were done to assess AURKA levels. The normalization of AURKA expression to the mitotic index of the tumor, was done by developing this IHC assay into a two color assay to also measure the mitotic marker phospho(Ser10) histone H3 (pHH3).
The Novocastra and Epitomics antibodies produced a specific band for AURKA and a proper modulation of AURKA when comparing asynchronous and nocodazole treated cells by Western blot analysis. The Novocastra antibody showed the best concordance by Western blot and IHC when comparing total AURKA expression in matched frozen and FFPE tumors. Further optimization of the total AURKA protein IHC assay was done using the Novocastra antibody. Testing of TMAs using this new assay demonstrated substantially lower percent positive AURKA expression compared to published reports: breast (35%), colon (32%), lung (39%), ovarian (12%), and prostate (7%). While there may be certain differences between the tumor indications tested here and those in the literature based on grade, stage and/or histological subtype, the IHC assay used here gives robust staining representative of total AURKA expression. Further, the AURKA protein level can now be normalized to the mitotic index by concurrently measuring the pHH3 level in tumors. This dual assay is now being used to assess AURKA protein expression and mitotic index in patient tumors from clinical studies with the first in class AURKA inhibitor alisertib.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A41.