A major function of long non-coding RNAs (lncRNAs) is regulating gene expression 
through changes in chromatin state. Experimental evidence suggests that in 
cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an 
occupancy pattern resembling that of the embryonic state. We have previously 
demonstrated that the expression level of lncRNA in the HOX locus, including 
HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA 
in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and 
ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in 
formalin-fixed paraffin-embedded breast cancer tissues in a high throughput 
tissue microarray format to correlate expression with clinicopathologic 
features. Though overall EZH2 and HOTAIR expression levels were highly 
correlated, the subset of cases with strong HOTAIR expression correlated with ER 
and PR positivity, while the subset of cases with strong EZH2 expression 
correlated with an increased proliferation rate, ER and PR negativity, HER2 
underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended 
with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and 
EZH2 had increased expression in the metastatic carcinomas. This is the first 
study to show that RNA in situ hybridization of formalin fixed paraffin-embedded 
clinical material can be used to measure levels of long non-coding RNAs. This 
approach offers a method to make observations on lncRNAs that may influence the 
cancer epigenome in a tissue-based technique.