Abstract P3-07-04: EphA2: An emerging target in triple-negative breast cancer
Authors: Eckhardt BL , Torres AM , Woodward WA , Krishnamurthy S , Meric-Bernstam F , Ueno NT 
Published: February 2018

Purpose: Breast tumors classified as 'triple negative' (TNBC) lack defining markers ER/PR/HER2 and do not have clinically-approved targeted therapy. This heterogeneous classification of breast cancers, while immediately responsive to standard chemotherapy, commonly develop resistance and have a poor five-year survival rate. As such, the identification of new therapeutic targets are warranted. As part of our drug discovery platform, we have identified EphA2, as a synthetic-lethal gene that enhances the therapeutic action of FDA-approved, anti-inflammatory compounds. Thus we sought to ascertain the relevance of EphA2-targeted therapy in TNBC, through the evaluation of the marker in preclinical and clinical specimens.

Methods: Sixty-one human and murine breast cancer cell lines or patient-derived xenografts were collated. Protein lysates were created from cells in vitro or from respective tumors established from cells implanted into NSG mice. Forty-nine tumors established (minimum 500mm3) and were surgically removed, fixed in formalin and paraffin embedded. A TMA was constructed with tumor specimens represented twice on the array and reflected all molecular subtypes including; ER-positive (n=5), PR-positive (n=3), HER2-positive (n=9) and TNBC (n=31). Immunostaining for EphA2 was performed with the rabbit monoclonal antibody EphA2 (D4A2) XP (Cell Signaling, #6997) using manufacturer's instructions. Immunostaining was evaluated using the H-score method (score between 0-300), with positive staining for EphA2 reflecting a score of 100 or greater. Analysis of breast cancer lysates by western blot was analyzed by absolute and relative quantitation methods; gene expression data was assessed through Oncomine or using the BreastMark algorithm (http://glados.ucd.ie/BreastMark/). This algorithm integrates gene expression and survival data from 26 datasets on 12 different microarray platforms corresponding to ˜17,000 genes in up to 4,738 samples.

Results: In an integrated gene expression platform (BreastMark), we observed that elevated EphA2 expression was associated with poor prognosis in a cohort of TNBC patient tumor samples. Western blot analysis of EphA2 protein on breast cancer cell lines, identified a greater percentage of TNBC cells expressing EphA2 compared to non-TNBC cell lines. EphA2 immunostaining was observed in the majority of tumor tissues. When present on cancer cells, EphA2 localized to the cell surface; while displaying ubiquitous localization within stromal populations. Cell surface expression of EphA2 on cancer cells was largely restricted to TNBC tumors (11/31 tumors, 35.5%) compared to other molecular subtypes (1/13 non-TNBC tumors, 7.7%; p = 0.0294). Expression of EphA2 in stromal cell populations was similar between groups (TNBC = 22/31, non-TNBC = 11/13; p = 0.1711).

Conclusions: Our analysis determined that EphA2 was specifically expressed on cancer cells derived from tumors with a 'triple-negative' molecular subtype. Collectively our data suggests that EphA2 is an emerging target in TNBC and that therapies directed against EphA2 may provide a significant benefit for a majority of patients that express this marker.

Citation Format: Eckhardt BL, Torres AM, Woodward WA, Krishnamurthy S, Meric-Bernstam F, Ueno NT. EphA2: An emerging target in triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-04.