Abstract 2962: LRH-1 expression in breast cancer tissue and its association with phenotype and DNA methylation
Authors: Pang Jia-Min , Chand Ashwini , Knower Kevin , Takano Elena , Byrne David , Sprenkeler Evelien , Molania Ramyar , Millar Ewan , Lee Soon , O'Toole Sandra , Clyne Colin , Dobrovic Alexander , Fox Stephen 
Published: August 2015

Abstract
Introduction
The LRH-1 gene (NR5A2) located on chromosome 1q codes for a transcription factor implicated in promoting cell proliferation and migration in breast cancer. The gene contains five CpG islands and codes for several mRNA isoforms. However, little is known about its expression in breast cancer tissues. In this study, we examine LRH-1 expression patterns in breast cancer tissue by immunohistochemistry, its association with phenotypic features, and the relationship of DNA methylation with LRH-1 expression.
Methods
LRH-1 immunohistochemical (IHC) staining was performed on tissue microarray sections containing 329 cases of breast cancer (175 ductal carcinoma in situ (DCIS), 154 invasive carcinomas). Methylation was assessed using methylation sensitive high resolution melting assays targeted towards regions in the second and fifth CpG islands of LRH-1. These CpG islands are situated immediately upstream of the transcription start sites of mRNA transcripts 1 and 3, respectively. CpG island 2 (CpG2) methylation was assessed in 96 cases (65 DCIS, 31 invasive carcinoma), methylation of CpG island 5 (CpG5) was assessed in 99 cases (66 DCIS, 33 invasive carcinoma), and methylation data for both CpG islands was obtained in 85 cases (55 DCIS, 30 invasive carcinoma).
Results
IHC staining revealed four patterns of nuclear staining in breast cancer tissues. Nuclear staining was present as either in a finely granular pattern (pattern 1), a sparse punctate pattern (pattern 2), a dense punctate pattern (pattern 2+), or as a coarse granular pattern (pattern 3). Patterns 1 and 2 were considered LRH-1 low and patterns 2+ and 3 were considered LRH-1 high. LRH-1 high staining was significantly associated with adverse phenotypic features including high grade (p&lt;0.0005), non-luminal phenotype (p = 0.003), estrogen receptor (ER) negativity (p = 0.008), progesterone receptor (PgR) negativity (p = 0.003) and HER2 amplification (p = 0.039). The presence of CpG5 methylation together with absent or low levels of CpG2 methylation was significantly associated with LRH-1-high IHC expression (p = 0.033) and aggressive phenotypic features of high grade (p = 0.001), non-luminal intrinsic subtype (p = 0.021), ER negativity (p = 0.010), PgR negativity (p = 0.021), and HER2 amplification (p = 0.043).
Conclusions
This study identifies, for the first time, distinct nuclear IHC staining patterns in breast cancer tissue and the association of LRH-1 IHC-high patterns with aggressive phenotypic features. Differential DNA methylation of two regions is associated with LRH-1 IHC staining pattern, indicating a role for DNA methylation in the control of differential mRNA isoform expression and thus protein expression.
Citation Format: Jia-Min Pang, Ashwini Chand, Kevin Knower, Elena Takano, David Byrne, Evelien Sprenkeler, Ramyar Molania, Ewan Millar, Soon Lee, Sandra O'Toole, Colin Clyne, Alexander Dobrovic, Stephen Fox. LRH-1 expression in breast cancer tissue and its association with phenotype and DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2962. doi:10.1158/1538-7445.AM2015-2962