Background: Clinical management of metastatic gastric cancer (mGC) remains a major challenge due to a lack of specific biomarkers and effective therapeutic targets. Recently, accumulating evidence has suggested that exosomes play an essential role in cancer metastasis and can be an excellent reservoir of novel biomarkers and candidate therapeutic targets for cancer. Therefore, in this study, we aimed to reveal the proteomic profile of mGC-derived exosomes. Methods: Exosomes were isolated from pooled serum samples of 20 mGC patients and 40 healthy controls (HC) by ultracentrifugation. Next, quantitative proteomic analyses were applied to analyze the protein profiles of the exosomes, and bioinformatic analyses were conducted on the proteomic data. Finally, the expression of exosomal protein candidates was selectively validated in individual subjects by western blot analysis. Results: We isolated exosomes from serum samples. The size of the serum derived exosomes ranged from 30 to 150 nm in diameter. The exosomal markers CD9 and CD81 were observed in the serum exosomes. However, the exosomal negative marker calnexin, an endoplasmic reticulum protein, was not detected in exosomes. Overall, 443 exosomal proteins, including 110 differentially expressed proteins (DEPs) were identified by quantitative proteomics analyses. The bioinformatics analyses indicated that the upregulated proteins were enriched in the process of protein metabolic, whereas the downregulated proteins were largely involved in cell-cell adhesion organization. Surprisingly, 10 highly vital proteins (UBA52,
Background: The research on circulating tumor DNA (ctDNA) in pancreatic cancer (PC) has emerged recently. Although the detection rate of the KRAS mutation in ctDNA was relatively consistent with that in tumor tissue, whether the KRAS mutant allele fraction (MAF) differed was still not reported. So far, the clinical application of ctDNA detection in PC remains inconclusive.Methods: Plasma samples were collected from 110 PC and 52 pancreatic benign (PB) disease patients. The detection of KRAS mutation in ctDNA was performed using droplet digital PCR and compared with that in matched tumor tissue. We assessed the utility of KRAS MAFs in ctDNA and tissue for pancreatic malignancy assessment.Results: We found that KRAS MAF in ctDNA of PC patients was higher than that of PB patients, and was obviously associated with tumor staging and distant metastasis. However, KRAS MAF in ctDNA was significantly different from that in matched tissue. KRAS MAF in tumor tissue had no significant correlation with the clinical status. In addition, a ROC curve analysis revealed that mutant KRAS ctDNA combined with CA19-9 could increase the sensitivity rate of early-stage PC prediction, compared with CA19-9 test alone.Conclusion: The MAF of KRAS in ctDNA was related to the clinical stage of PC (p = 0.001). Mutant KRAS ctDNA could improve the sensitivity in early diagnosis of PC as a complement to CA19-9. Our study suggested that KRAS mutation in ctDNA could be a valuable circulating biomarker for the malignancy assessment in PC.
Background: Recently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application. Methods: Taking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis. Results: We found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number. Conclusion: This study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.
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