Research Article: Clinical significance of glypican-3-positive circulating tumor cells of hepatocellular carcinoma patients: A prospective study

Date Published: May 29, 2019

Publisher: Public Library of Science

Author(s): Michinori Hamaoka, Tsuyoshi Kobayashi, Yuka Tanaka, Hiroaki Mashima, Hideki Ohdan, Matias A Avila.


The utility of glypican-3 (GPC3) expression for the detection of circulating tumor cells (CTCs) in hepatocellular carcinoma (HCC) patients has not been elucidated. The aim of this study was to identify associations between the presence of GPC3-positive CTCs and clinicopathological factors of these patients, furthermore, to evaluate whether CTC can predict microscopic portal vein invasion (mPVI). This study was done on 85 patients who underwent hepatectomy as the first-line treatment and whose preoperative imaging showed no evidence of macroscopic PVI and distant metastases. Peripheral blood was collected from all patients immediately before surgery. Cells were purified initially by density gradient centrifugation followed by immunomagnetic positive enrichment based upon the expression of GPC3. The numbers of CTCs contained in the enriched samples were enumerated via flow cytometry. Protocol validation using HepG2 cells spiked into 8.0 mL of blood from a healthy volunteer indicated that we were able to recover 12.1% of the tumor cells. A median number of 3 CTCs (range: 0–27) was detected in the 8.0 mL of peripheral blood of the 85 analyzed HCC patients. Thirty-three patients had CTCs ≥5, and these patients had a higher incidence of mPVI (p < 0.001), a lower disease-free survival (p = 0.015), and a lower overall survival (p = 0.047) than those with CTCs <5. Multivariate analysis identified CTCs ≥5 as an independent predictor of mPVI (p < 0.001). In conclusion, preoperative GPC3-positive CTCs ≥5 was a risk factor of mPVI and poor prognosis, and therefore may be a useful biomarker for HCC patient outcomes.

Partial Text

Although hepatectomy is expected to be curative for hepatocellular carcinoma (HCC), tumor recurrence, most of which occurs in the remnant liver, is common in these patients. Indeed, approximately 30% of these patients develop recurrence within the first year after surgery, and early recurrence is one of the most important prognostic factors for this disease [1]. Tumor invasion in the portal vein, even if it is microscopic portal vein invasion (mPVI), is associated with increased risk leading to early recurrences. Although anatomical liver resection should be considered for the patients with mPVI, it is difficult to detect mPVI before the treatment of HCC [2–5]. Therefore, more reliable marker of mPVI is needed.

In the present study, CTCs were enumerated via flow cytometry after density gradient centrifugation and immuno-magnetic positive enrichment based upon expression of GPC3 in HCC patients. The present study found that patients in the GPC3-positive CTC ≥5 group had a higher incidence of mPVI, and a lower disease-free survival, and a lower overall survival than those in the GPC3-positive CTC <5 group. Multivariate analysis identified GPC3-positive CTCs ≥5 as an independent predictor of mPVI. To the best of our knowledge, this is the first study on positive enrichment based upon the expression of GPC3, and also the first study of the predictor for mPVI using GPC3-positive CTCs.   Source:


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