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Expression of Glypican-3 is an independent prognostic biomarker in primary gastric cancer and corresponding serum exosomes
13 August 2018 (online)
Exosomes are nano-sized membranous vesicles of endosomal origin and released by cancer cells for the transfer RNA, DNA and proteins of their cellular origin. Exosomes have therefore gained increasing attention as non-invasive biomarkers in cancer. The expression of Glypcian-3 (GPC-3), a Heparane-Sulfate-Proteoglycan (HSPG), has been associated with varying survival outcomes in gastric cancer (GC). The value of exosomal Glypican-3 (eGPC-3) and tissue based Glypican-3 (tGPC-3) as diagnostic and prognostic biomarkers in patients with GC has not been investigated yet.
Exosomes were isolated from serum samples of patients with GC or adenocarcinoma of the esophagogastric junction (AEG) (n = 49) who underwent surgical resection and from control subjects (n = 56) with no evidence of neoplastic disease. The detection of exosomes was confirmed by nanoparticle-tracking analysis (NTA), transmission electron microscope (TEM), western blotting (CD9, CD63 and TSG101) and fluorescent nanoparticle-tracking analysis (fNTA) (CD9, CD63 and TSG101) in representative samples. Subsequently, the expression of eGPC-3 was assessed by flow cytometry as well as the expression of GPC-3 on gastric cancer tissue specimens by immunohistochemistry (IHC). Furthermore, expression of tGPC-3 has been correlated to clinicopathologic parameters and validated in two independent external control populations.
The presence of exosomes could be confirmed by NTA, TEM, western blotting and fNTA. We show that GPC-3 is significantly decreased on serum exosomes from gastric cancer patients compared to healthy donors (p < 0.0001). ROC curve analysis resulted in an AUC of 0.85 when comparing eGPC-3 of gastric cancer patients with healthy donors, outperforming current standard biomarkers (CEA, CA 19 – 9 and CA 72 – 4).
On univariate analysis, low eGPC-3 and high tGPC-3 expression was associated significantly with poor overall survival (p = 0.041). High expression of tGPC-3 was validated as strong prognostic factor in two independent cohorts. Cox regressional analysis significantly confirmed tGPC-3 as an independent prognostic marker (p = 0.02).
Our study for the first time suggests eGPC-3 and tGPC-3 as potential diagnostic and prognostic biomarkers for GC and AEG.