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      Plasma microRNA Levels Combined with CEA and CA19-9 in the Follow-Up of Colorectal Cancer Patients

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          Abstract

          Colorectal cancer (CRC) ranks among the most common cancers worldwide. Surgical removal remains the best strategy for treatment of resectable tumors. An important part of caring for patients after surgery is monitoring for early detection of a possible relapse of the disease. Efforts are being made to improve the sensitivity and specificity of routinely used carcinoembryonic antigen (CEA) with the use of additional biomarkers such as microRNAs. The aim of our study was to evaluate the prognostic potential of microRNAs and their use as markers of disease recurrence. The quantitative estimation of CEA, CA19-9, and 22 selected microRNAs (TaqMan Advanced miRNA Assays) was performed in 85 paired (preoperative and postoperative) blood plasma samples of CRC patients and in samples taken during the follow-up period. We have revealed a statistically significant decrease in plasma levels for miR-20a, miR-23a, miR-210, and miR-223a ( p = 0.0093, p = 0.0013, p = 0.0392, and p = 0.0214, respectively) after surgical removal of the tumor tissue. A statistically significant relation to prognosis (overall survival; OS) was recorded for preoperative plasma levels of miR-20a, miR-21, and miR-23a ( p = 0.0236, p = 0.0316, and p =0.0271, respectively) in a subgroup of patients who underwent palliative surgery. The best discrimination between patients with favorable and unfavorable outcomes was achieved by a combination of CEA, CA19-9 with miR-21, miR-20a, and miR-23a ( p < 0.0001). The use of these microRNAs for early disease recurrence detection was affected by a low specificity in comparison with CEA and CA19-9. CEA and CA19-9 had high specificity but low sensitivity. Our results show the benefit of combining currently used standard biomarkers and microRNAs for precise prognosis estimation.

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          Circulating Exosomal microRNAs as Biomarkers of Colon Cancer

          Purpose Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. Experimental Design Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. Results The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. Conclusion Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.
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            Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer.

            The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC). To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided. Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03). Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.
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              Tumour markers in colorectal cancer: European Group on Tumour Markers (EGTM) guidelines for clinical use.

              The aim of this article is to present updated guidelines for the use of serum, tissue and faecal markers in colorectal cancer (CRC). Lack of specificity and sensitivity preclude the use of all existing serum markers for the early detection of CRC. For patients with stage II or stage III CRC who may be candidates for either liver resection or systemic treatment should recurrence develop, CEA should be measured every 2-3 months for at least 3 years after diagnosis. Insufficient evidence exists to recommend routine use of tissue factors such as thymidylate synthase, microsatellite instability (MSI), p53, K-ras and deleted in colon cancer (DCC) for either determining prognosis or predicting response to therapy in patients with CRC. Microsatellite instability, however, may be used as a pre-screen for patients with suspected hereditary non-polyposis colorectal cancer. Faecal occult blood testing but not faecal DNA markers may be used to screen asymptomatic subjects 50 years or older for early CRC.
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                Author and article information

                Journal
                Cancers (Basel)
                Cancers (Basel)
                cancers
                Cancers
                MDPI
                2072-6694
                21 June 2019
                June 2019
                : 11
                : 6
                : 864
                Affiliations
                [1 ]Department of Biology, Faculty of Medicine in Pilsen, Charles University, alej Svobody 76, 32300 Pilsen, Czech Republic; martin.pesta@ 123456lfp.cuni.cz (M.P.); katerina.houfkova@ 123456lfp.cuni.cz (K.H.); tereza.macanova@ 123456lfp.cuni.cz (T.M.)
                [2 ]Laboratory of Immunoanalysis, University Hospital in Pilsen, E. Benese 13, 30599 Pilsen, Czech Republic; kucerar@ 123456fnplzen.cz (R.K.); topolcan@ 123456fnplzen.cz (O.T.); karlikovam@ 123456fnplzen.cz (M.K.); jiri.polivka@ 123456lfp.cuni.cz (J.P.); iva.machova@ 123456lfp.cuni.cz (I.M.); slouka@ 123456fnplzen.cz (D.S.)
                [3 ]Department of Medical Chemistry and Biochemistry, Faculty of Medicine in Pilsen, Charles University, Karlovarska 48, 30166 Pilsen, Czech Republic
                [4 ]Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Karlovarska 48, 30166 Pilsen, Czech Republic
                Author notes
                [* ]Correspondence: vlastimil.kulda@ 123456lfp.cuni.cz ; Tel.: +420-377-593-287
                Author information
                https://orcid.org/0000-0003-3231-4509
                https://orcid.org/0000-0002-3988-7898
                https://orcid.org/0000-0001-7926-994X
                Article
                cancers-11-00864
                10.3390/cancers11060864
                6627112
                31234350
                1aaf45f7-6b44-42bb-a069-843c0a4c08bd
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 14 May 2019
                : 19 June 2019
                Categories
                Article

                colorectal cancer,biomarkers,recurrence,microrna,cea,ca19-9,blood plasma

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