Abstract 3465: microRNA profiling of breast ductal lavage fluid.
Authors: do Canto Luisa Matos , Willey Shawna C. , Feldman Elizabeth D. , Li Xin (James) , Marian Catalin , Haddad Bassem R. 
Published: August 2014

Abstract
Introduction: MicroRNAs (miRNAs) have been detected in tumors as well as in serum, plasma, and other body fluids. We are investigating whether we can detect miRNAs in the breast ductal lavage (DL) fluid and whether miRNA profiling of the fluid can be used as a marker for early detection of breast cancer (BC). Emerging data are showing that miRNAs have a great potential as cancer biomarkers: miRNAs are aberrant in cancers, exceptionally stable in various biological specimens, and are tissue specific.
Approach: We are evaluating the miRNA profiles in DL fluid samples and primary tumors that have been obtained from 22 patients with DCIS and Invasive BC. The DL samples were collected in the operating room from patients with confirmed diagnosis, prior to their surgery. For each patient, two DL samples were obtained: one from the affected breast and the other from the contralateral normal breast (control). Each patient served as her own control. Post surgery, a primary tumor specimen was obtained. Total RNA was extracted from 250ul ductal lavage samples and from primary tumors using the Qiagen miRNeasy kit and the Ambion RecoverALL Total Nucleic Acid Isolation Kit for FFPE, respectively, and the quantity of RNA was assessed using a Thermo Scientific NanoDropTM Spectrophotometer. Micro RNA profiling was done using the TaqMan(R) Human MicroRNA Array Set v3.0 (Applied Biosystems), a quantitative real time PCR based array containing 742 human miRNAs, 3 endogenous controls to aid in data normalization and one assay not related to humans as a negative control. Integromics StatMiner was used to analyze the data, and 2ˆ-ddCt method was applied. The data were first cleaned by filtering out those miRNAs with 75% of samples having Ct values more than 36. Thereafter cleaned Ct values were normalized to the reference control miRNA to get deltaCt values, and then normalized by median center method. Paired t-test was used to test the significance of three comparisons, DL from cancerous breast (LT) vs. DL from normal breast (LC), primary tumor (PT) vs. LC and PT vs. LT, each comparison resulted in a miRNA list
Results: To date, we completed the analysis of samples from 12 of the 22 study subjects. For each subject we evaluated 2 DL samples and a primary tumor sample, 36 samples in total, to identify significantly differentially expressed miRNAs. Based on the analysis, we identified 2 miRNAs that discriminated between the DL from the cancerous breast vs. the normal control. These 2 miRNAs are also differentially expressed in the primary tumors compared to the control fluid.
Conclusion: Our data show that miRNA analysis is feasible in breast ductal lavage fluid. Furthermore, two miRNAs have been identified to be differentially expressed in the DL fluid from affected breasts. Our findings suggest that miRNA analysis is potentially useful for detection of BC using ductal fluid analysis and justify a larger study to confirm these preliminary results.
Supported by a Grant from the Avon Foundation for Women
Citation Format: Luisa Matos do Canto, Shawna C. Willey, Elizabeth D. Feldman, Xin (James) Li, Catalin Marian, Bassem R. Haddad. microRNA profiling of breast ductal lavage fluid. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2013-3465