Abstract P4-08-02: Understanding the mechanisms of action underlying the role of IL6ST, a key biomarker for prediction of response to endocrine therapy
Authors: Turnbull AK , Fernando A , Martinez-Perez C , Finch AJ , von Kriegsheim A , Wills J , Quinn N , Selli C , Mosley D , Langdon SP , Sims AH , Dixon JM 
Published: February 2018

Introduction: IL6ST is regarded as a putative ER target gene. Recently it has been recognised as a key biomarker for prediction of response to endocrine therapy (ET), having been included as the primary biomarker in our EA2Clin test and as an ER-signalling gene in the EndoPredict test. In both tests higher IL6ST expression is associated with a better response to ET and better prognosis. Despite its importance as a biomarker, little is known about its functional role in breast cancer (BC).

Methods: Pre- and on-treatment (at 14-days and at surgery) samples were collected from 102 post-menopausal women with ER+ BC, treated with 3-6 months of neoadjuvant ET. RNA was extracted for whole-genome expression analysis. From a subset with available fresh frozen tissue (28 patients, 83 samples) protein was extracted and proteome analysis using mass spectrometry is currently underway – results available for SABCS 2017. Immunohistochemistry was performed on FFPE tissue microarrays (TMAs) comprising pre-treatment samples from 102 patients. Cytoplasmic/membrane staining was scored using a graduated scale (0-3+) and nuclear staining was graded using an Immunoscore.

Results: IL6ST exists in membrane-bound and soluble forms of varying size. The full-length membrane bound molecule comprises 8 domains: 6 extracellular, 1 transmembrane and 1 cytoplasmic. In the EA2Clin test, pre-treatment BC tissues are stained for IL6ST with an antibody specific for a region spanning the transmembrane and cytoplasmic domains. TMAs were stained for IL6ST with both this and a second antibody binding the extracellular part, detecting both full-length and most soluble isoforms. Levels of both were correlated (R=0.82, P&lt;0.0001).

IL6ST is known to mediate the action of cytokines including IL6, OSM and LIF via downstream regulation of pathways such as JAK/STAT. TMAs were stained for antibodies against IL6ST, OSM, IL6, total STAT3, pSTAT3 (Tyr705) and pSTAT3 (Ser727). IL6ST was scored as low (0/1+) or high (2+/3+). There was a positive association between levels of IL6ST and IL6 (P=0.02) and total STAT3 (P=0.003). There was no association between IL6ST and OSM or either pSTAT3.

Supervised gene expression analysis comparing pre-treatment samples with high and low IL6ST levels revealed increased levels of STAT3-regulated genes: cell cycle (CEBPD, CDKN1B), apoptosis (NFIL3, ATF3, BCL2), extracellular matrix remodelling (ADM, SEPRINE1-3) and interferon signalling (IFIT1, IFI44, IFI27). Unsupervised gene enrichment analysis revealed increased expression of genes involved with JAK/STAT, PI3K, mTOR and ERBB1 signalling in tumours expressing higher IL6ST levels. Lower levels were associated with increased energy generation, cellular metabolism and epithelial-mesenchymal transition.

Conclusions:

• This is the first matched whole-genome and mass spectrometry proteome analysis of sequential ET-treated BC patients

• IL6ST predicts response to ET – it is used in2 independent assays

• Levels of full-length IL6ST appear to be the most important for ET response prediction

• IL6ST may have an active role in BC cells, mediating signalling of cytokines such as IL6 through the JAK/STAT pathway and subsequent downstream transcriptional regulation.

Citation Format: Turnbull AK, Fernando A, Martinez-Perez C, Finch AJ, von Kriegsheim A, Wills J, Quinn N, Selli C, Mosley D, Langdon SP, Sims AH, Dixon JM. Understanding the mechanisms of action underlying the role of IL6ST, a key biomarker for prediction of response to endocrine therapy [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 P4-08-02.