Abstract 1498: Long non-coding RNA in situ hybridization signal patterns correlate with breast tumor pathology
Authors: Zhang Zhouwei , Peng Zhihua , Olsen Daniel , deKay James , Weaver Donald L. , Evans Mark F. 
Published: May 2015

Abstract
Background: Long non-coding RNAs (lncRNAs) participate in a spectrum of biological activities by diverse mechanisms. LncRNA dysregulation has been reported for many cancers. Some lncRNAs may function as oncogenes (OG) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR. In this study we have examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH).
Methods: Expression of six lncRNAs, HOTAIR (OG), H19 (OG), Kcnq1ot1 (OG), Meg3 (TSG), Malat1 (OG), and ZFas1 (TSG), plus HER2 and MKI67 mRNAs was examined by RNAscope® CISH using tissue microarrays (TMAs) comprising normal epithelia, ductal carcinoma in situ (DCIS), and invasive carcinoma (IC) from 45 patients. HOTAIR/H19-mediated protein EZH2 was evaluated by immunohistochemistry (IHC). CISH and IHC results were scored in terms of the percentage of stained cells (&lt;2%=0, 2-10%=1, 10-50%=2, &gt;50%=3) and staining intensity (low=1, medium=2, high=3). Staining grade (SG) values were calculated as SG= proportion x intensity (SG value range=1-9).
Results: The TMAs contained 36 normal epithelia (N), 34 DCIS (D) and 43 IC tissue punches.
HOTAIR expression was common: at SG≥1, expression was detected in N:85%; D:97%; IC:100% (p&gt;0.05). HOTAIR SG mean values (N 4.0, D 5.7, IC 6.2) were significantly higher in DCIS and IC than in normal tissues (N:D p&lt;0.05; N:IC p&lt;0.01; D:IC p&gt;0.05). IC HOTAIR SGs correlated with Nottingham grade (p&lt;0.01), HER2 CISH (p&lt;0.01) and MKI67 CISH (p&lt;0.05). HOTAIR expression levels in DCIS were associated with MKI67 CISH (p&lt;0.01).
H19 was rarely expressed in normal epithelial or tumor cells but was strongly expressed especially in inter-lobular stromal cells around invasive growths. At SG≥1 H19 was detected in N:38%; D:55%; IC:90% (p&lt;0.01). H19 SG means (N 1.3, D 2.3, IC 3.7) were significantly higher among IC than normal or DCIS tissues (N:D p&gt;0.05; N:IC p&lt;0.01; D:IC p&lt;0.05). IC H19 expression showed a positive correlation with Nottingham grade (p&lt;0.05) and with MKI67 expression (p&lt;0.01). H19 in DCIS showed a correlation with MKI67 CISH (p&lt;0.01).
Kcnq1ot1 staining was more common in DCIS and IC than in normal tissues: at SG≥1, N:77%; D:96%; IC:94% (p&lt;0.01). Both the DCIS and IC SGs correlated with MKI67 expression (p&lt;0.05). Meg3 expression was associated with normal breast; at SG≥1, N:46%; D:2%; IC:0%, [p&lt;0.01]). Malat1 stained strongly in all cells in all specimens. Zfas1 specimen staining was absent or weak.
IC and DCIS EZH2 IHC staining correlated positively with both HOTAIR and H19 expression (p&lt;0.05).
Conclusions: These data demonstrate the utility of CISH for investigating and demonstrating pathologic lncRNA expression. HOTAIR and H19 in particular and possibly Kncq1ot1 and Meg3 are potential breast cancer biomarkers. H19 may be a marker for DCIS at increased risk of progression to invasive cancer.
Citation Format: Zhouwei Zhang, Zhihua Peng, Daniel Olsen, James deKay, Donald L. Weaver, Mark F. Evans. Long non-coding RNA in situ hybridization signal patterns correlate with breast tumor pathology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1498. doi:10.1158/1538-7445.AM2014-1498