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Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA.

ABSTRACT: Serum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The association of a fraction of various serum proteins such as carcinoembryonic antigen (CEA) with circulating exosomes has been debated. The establishment of a method to measure the exosomal fraction of such proteins might help resolve this controversy. The use of enzyme-linked immunosorbent assays (ELISAs) to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. In this study, we optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA.One hundred sixteen consecutive patients with colorectal cancer were enrolled for this study between June 2015 and June 2016 at Wakayama Medical University Hospital (WMUH). Whole blood samples were collected from patients during surgery. Exosomes were isolated using the ExoQuick reagent, solubilized in an assay buffer and subjected to CEA detection by ELISA. The procedure of serum exosome isolation and the formulation of the assay buffer used for the ELISA were optimized in order to improve the sensitivity and specificity of the assay.A five-fold increase in the concentration of the exosomes in the assay buffer (using initial serum volume as a reference) and the addition of bovine serum albumin (BSA) resulted in more accurate measurements of the serum exosomal CEA. The thawing temperature of frozen serum samples before exosome extraction was also optimized. A validation study that included one hundred sixteen patients with colorectal cancer demonstrated that serum exosomal CEA from samples thawed at 25°C exhibited a better AUC value, sensitivity, and specificity as well as a more correct classification than serum CEA.We optimized an easy and rapid detection method for assessment of serum exosomal CEA. The thawing temperature of frozen serum prior to exosome extraction, the formulation of the assay buffer used for exosome solubilization and the concentration of the exosomes in this buffer were fine-tuned to enable the appropriate and accurate measurement of serum exosomal CEA.

SUBMITTER: Yokoyama S

PROVIDER: S-EPMC5560664 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:ImportanceGuidelines recommend measuring preoperative carcinoembryonic antigen (CEA) in patients with colon cancer. Although persistently elevated CEA after surgery has been associated with increased risk for metastatic disease, prognostic significance of elevated preoperative CEA that normalized after resection is unknown.ObjectiveTo investigate whether patients with elevated preoperative CEA that normalizes after colon cancer resection have a higher risk of recurrence than patients with normal preoperative CEA.Design, setting, and participantsThis retrospective cohort analysis was conducted at a comprehensive cancer center. Consecutive patients with colon cancer who underwent curative resection for stage I to III colon adenocarcinoma at the center from January 2007 to December 2014 were identified.ExposuresPatients were grouped into 3 cohorts: normal preoperative CEA, elevated preoperative but normalized postoperative CEA, and elevated preoperative and postoperative CEA.Main outcomes and measuresThree-year recurrence-free survival (RFS) and hazard function curves over time were analyzed.ResultsA total of 1027 patients (461 [50.4%] male; median [IQR] age, 64 [53-75] years) were identified. Patients with normal preoperative CEA had 7.4% higher 3-year RFS (n = 715 [89.7%]) than the combined cohorts with elevated preoperative CEA (n = 312 [82.3%]) (P = .01) but had RFS similar to that of patients with normalized postoperative CEA (n = 142 [87.9%]) (P = .86). Patients with elevated postoperative CEA had 14.9% lower RFS (n = 57 [74.5%]) than the combined cohorts with normal postoperative CEA (n = 857 [89.4%]) (P = .001). The hazard function of recurrence for elevated postoperative CEA peaked earlier than for the other cohorts. Multivariate analyses confirmed that elevated postoperative CEA (hazard ratio [HR], 2.0; 95% CI, 1.1-3.5), but not normalized postoperative CEA (HR, 0.77; 95% CI, 0.45-1.30), was independently associated with shorter RFS.Conclusions and relevanceElevated preoperative CEA that normalizes after resection is not an indicator of poor prognosis. Routine measurement of postoperative, rather than preoperative, CEA is warranted. Patients with elevated postoperative CEA are at increased risk for recurrence, especially within the first 12 months after surgery.

Project description:Background/Aim: We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Results: NGS data revealed that exosomal miR-660-5p, miR-125a-5p, and miR-122 expression were changed significantly with the progression of liver fibrosis, of which miR-122 exhibited high read counts enough to be used as a biomarker. The level of exosomal miR-122 decreased as the pathologic fibrosis grade progressed and patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P < 0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis especially in combination with fibrosis-4 score and transient elastography. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker was greatly improved. Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of various fibrosis related proteins. Conclusion: Exosomal miR-122 may serve as a useful non-invasive biomarker for liver fibrosis, especially in patients with non-viral etiologies of chronic liver disease.

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Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA.

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