Abstract 5187: Expression of metastasis-related miRNAs in EpCAM-positive CTC fraction in corresponding plasma and FFPEs of breast cancer patients with verified metastasis

Introduction: CTC play a critical role in the metastatic spread of carcinomas and their detection is associated with prognosis in many human cancers while their enumeration has been cleared by the FDA for follow up of breast, colon, and prostate cancer patients. However, it is quite clear now that simple enumeration of CTC is not enough and that CTC molecular characterization is absolutely necessary, since it can play a crucial role in understanding the biology of metastasis and in selecting patients for targeted therapy. In the present study, we investigated for the first time the expression levels of five metastasis-related miRNAs in the EpCAM-positive CTC fraction, in corresponding plasma and paired FFPEs samples of metastatic breast cancer (MBC) patients. Materials and Methods: We studied the expression of miR-21, miR-31, miR-146a, miR-200c and miR-210 expression in the EpCAM-positive CTC fraction and corresponding plasma samples and FFPEs tissues of 22 breast cancer patients with verified metastasis and 20 healthy individuals. For CTC analysis 20mL peripheral blood were used, RNA was extracted from the EpCAM positive CTC fraction, while circulating miRNAs were isolated from 200μL of corresponding plasma samples. miRNA quantitative analysis in both EpCAM-positive fractions and in corresponding plasma was performed by a stem-loop cDNA approach and RT-qPCR in the LightCycler 2.0 (Roche, Germany) Results: We first evaluated the differences in the expression levels of these five metastasis-related miRNAs between primary breast cancer tissues (FFPEs) and non-camcerous breast tissues (mammoplasties). The expression of all miRNAs was significantly different in primary tumors in respect to cancer-free breast tissues. Based on this finding we further studied the expression levels of these miRNAs in the EpCAM-positive CTC fraction and plasma samples of metastatic breast cancer in respect to healthy donors and found that they were also significantly different. There was a concordance only between miR-21 expression in EpCAM positive CTC fraction and FFPEs for 16/22 (72.7%) of patients (P = 0.026, Pearson's χ2 test), but not for the other miRNAs tested. The expression levels of all miRNAs in the EpCAM positive CTC fraction and circulating miRNAs were not associated with any of the clinical and pathological characteristics of the patients. We found only a correlation between circulating miR-210 expression levels in plasma and expression of ER (P = 0.043) and progest