Investigation of DNA and RNA markers by novel technologies demonstrates DNA content intratumoral heterogeneity and long non-coding RNA aberrations in breast tumors

摘要

BACKGROUND:Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50µm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH).METHODS:Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests.RESULTS:Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (pCONCLUSION:Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.